Payload-supported multispecific antibodies

Abstract

This disclosure relates to a modification of ForCE technology (e.g., described in Denglet al. 2020 and WO2019 / 077092 A1) that can be used in the production of payload-carrying molecules (such as antibody-drug conjugates) by combining a functional (e.g., binding) entity with a payload coupling Fc molecule. The principle on which this disclosure is based is shown in the schematic diagram in Figure 1.

Description

[Technical Field]

[0001] This disclosure relates to the field of molecular biology, particularly to antigen-binding molecular technology. [Background technology]

[0002] Antibody derivatives with attached payloads (e.g., antibody-drug conjugates, ADCs) serve as therapeutic agents, diagnostic agents, and research tools. For these purposes, ADCs must retain preferentially unimpaired antigen binding, as well as the desired payload function and potency (Nath et al. 2016; Akkapeddi et al. 2016). ADCs applied as drugs must have a more defined composition. This is particularly challenging for first-generation conjugates, which have payloads attached via NHS chemistry to free amines (lysine residues) exposed on the antibody surface. Amine-exposed lysine modified by the NHS is not only scattered across the antibody surface; in some cases, it is also present on either the antibody's CDR or adjacent Fv framework. Conjugation on or near the CDR can impair binding to the target antigen (Nath et al. 2016; Sadiki et al. 2020). Another obstacle to ADC discovery and screening approaches, including functional ranking, is the difficulty in ensuring identical or at least functionally equivalent conjugations of payloads to different antibodies.

[0003] Problems associated with random coupling can be addressed to some extent by applying site-directed conjugation. Most such approaches are based on the introduction of mutant residues that are targets of site-directed coupling. Examples include thiomab with exposed cysteine ​​(Akkapeddi et al. 2016), antibodies with modified amino acids introduced during translation (e.g., via stop codon repression (Beck et al. 2017; Patterson et al. 2014)), and antibodies with "tags" that enable spontaneous or enzyme-based conjugation (e.g., intein, SNAP, saltase, transglutaminase (Beck et al. 2017; Hussain et al. 2021; Mohlmann et al. 2011; Steffen et al. 2017)). Production and scaling of these techniques remain complex and cumbersome in the early project stages (or screening), as each conjugate must be produced and carefully analyzed individually.

[0004] Further challenges remain. One major bottleneck is the fact that ADCs with desired functionality are not simply the result of coupling the desired payload (e.g., cytotoxin) to an antibody that functions well at a site-specific location. Instead, the binding module (antibody, paratope, format) must be compatible with the payload, and the mode of attachment (linker composition) and location must be compatible with the binder and payload functionality. Stoichiometry (i.e., how many payloads are coupled to the antibody at which locations (drug-to-antibody ratio; DAR)) also affects ADC functionality and often modulates biophysical and pharmacokinetic properties (Beck et al. 2017; Sun et al. 2017). Therefore, the generation and identification / selection of optimal ADCs requires combinations of different binders and formats, or combinations with various linker-payload modules of different locations and different DARs. Thus, finding the optimal ADC requires evaluation of a matrix of combined parameters. Even with a small number of variables for each parameter (binder, format, linker, payload, position, DAR), the matrix becomes large. Producing a comprehensive ADC matrix to cover that design space is cumbersome and a major obstacle in early development (e.g., read identification).

[0005] The use of chain exchange-based format chain exchange (ForCE) technology to generate large binder-format bispecific antibody (bsAb) matrices has recently been described, for example, in Dengl et al. 2020 and International Publication No. 2019 / 077092 A1. ForCE is efficient, high-throughput, and automated-compatible, producing bispecific antibody combinations in a different format from monospecific input molecules in vitro. The precursor molecule is applied as an input module, which is a half-antibody complemented by a dummy, both related to a partially destabilized CH3 interface. The complementary precursor combination triggers an exchange reaction that produces a bsAb binder-binder-positional-stoichiometric matrix (Dengl et al. 2020). [Overview of the project]

[0006] In a first aspect, the present disclosure relates to a method for producing polypeptide complexes, below: (1) A first polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; The first polypeptide complex is formed by an interaction between the first polypeptide and the second polypeptide, and this interaction involves the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide contains a knob modification, and the CH3 domain of the second polypeptide contains a hole modification; The CH3 domain of the first polypeptide or the CH3 domain of the second polypeptide includes destabilizing modifications to destabilize the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The first polypeptide and / or the second polypeptide further comprises the first polypeptide complex, which includes a payload portion; (2) A second polypeptide complex comprising a third polypeptide containing a CH3 domain and a fourth polypeptide containing a CH3 domain; The second polypeptide complex is formed by the interaction between a third polypeptide and a fourth polypeptide, and the interaction includes the association between the CH3 domain of the third polypeptide and the CH3 domain of the fourth polypeptide; The CH3 domain of the third polypeptide includes a knob modification, and the CH3 domain of the fourth polypeptide includes a hole modification; Incubate the second polypeptide complex with the CH3 domain of the third polypeptide or the CH3 domain of the fourth polypeptide, which includes a destabilizing modification for destabilizing the association between the CH3 domain of the third polypeptide and the CH3 domain of the fourth polypeptide; Forming a third polypeptide complex comprising the first polypeptide and the fourth polypeptide, and / or a fourth polypeptide complex comprising the second polypeptide and the third polypeptide; and Recovering the third polypeptide complex and / or the fourth polypeptide complex; including The destabilizing modification of the CH3 domain of the first polypeptide or the second polypeptide does not destabilize the association between the first polypeptide and the fourth polypeptide, nor does it destabilize the association between the second polypeptide and the third polypeptide; The destabilizing modification of the CH3 domain of the third polypeptide or the fourth polypeptide does not destabilize the association between the first polypeptide and the fourth polypeptide, nor does it destabilize the association between the second polypeptide and the third polypeptide, providing a method.

[0007] In some embodiments, the destabilizing modification of the CHS domain of the first polypeptide or the second polypeptide stabilizes the association between the CH3 domain of the first polypeptide and the CH3 domain of the fourth polypeptide, and / or stabilizes the association between the CH3 domain of the second polypeptide and the CH3 domain of the third polypeptide.

[0008] In some embodiments, destabilizing modifications to the CH3 domain of the third polypeptide or the fourth polypeptide stabilize the association between the CH3 domain of the first polypeptide and the CH3 domain of the fourth polypeptide and / or stabilize the association between the CH3 domain of the second polypeptide and the CH3 domain of the third polypeptide.

[0009] In some embodiments, the first polypeptide includes a payload portion and the fourth polypeptide includes a functional portion, or the second polypeptide includes a payload portion and the third polypeptide includes a functional portion.

[0010] In some embodiments, (a) the CH3 domain of the first polypeptide includes 370E, the CH3 domain of the fourth polypeptide includes 357K; optionally, the CH3 domain of the second polypeptide includes 357E and the CH3 domain of the third polypeptide includes 370K; or (b) the CH3 domain of the first polypeptide includes 370K, the CH3 domain of the fourth polypeptide includes 357E; optionally, the CH3 domain of the second polypeptide includes 357K and the CH3 domain of the third polypeptide includes 370E.

[0011] In some embodiments, (a) the CH3 domain of the first polypeptide includes 366W and 370E, the CH3 domain of the second polypeptide includes 407V, 366S and 368A, the CH3 domain of the third polypeptide includes 366W, and the CH3 domain of the fourth polypeptide includes 407V, 366S, 368A and 357K; or (b) the CH3 domain of the first polypeptide includes 366W, 370E and 354C, the CH3 domain of the second polypeptide includes 407V, 366S and 368A, the CH3 domain of the third polypeptide includes 366W, and the CH3 domain of the fourth polypeptide includes 407V, 366S, 368A, 357K and 349C; or (c) The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 24, the CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 49, the CH3 domain of the third polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 48, and the CH3 domain of the fourth polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 25; or (d) The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 26, the CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 49, the CH3 domain of the third polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 48, and the CH3 domain of the fourth polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 27.

[0012] In some embodiments, the payload portion is or includes a detectable portion, a fluorescent portion, a luminescent portion, a radiopaque / contrast agent, a radiolabel, an immunodetectable portion, a portion having detectable activity, an enzyme portion, a drug portion, or a cytotoxic portion.

[0013] In some embodiments, the functional portion is or comprises a binding portion, an antibody or its target-binding fragment or derivative, a target-binding peptide / polypeptide, a target-binding nucleic acid, a detectable portion, a fluorescent portion, a luminescent portion, a radiopaque / contrast agent, a radiolabel, an immunodetectable portion, a portion having detectable activity, an enzyme portion, a drug portion, or a cytotoxic portion.

[0014] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, and / or the fourth polypeptide further include a CH2 domain and / or a hinge region.

[0015] The disclosure also provides polypeptide complexes comprising a third polypeptide complex or a fourth polypeptide complex produced by the method relating to the disclosure.

[0016] This disclosure also relates to a polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide contains a knob modification, and the CH3 domain of the second polypeptide contains a hole modification; The CH3 domain of the first polypeptide contains a destabilizing modification, and the CH3 domain of the second polypeptide contains a destabilizing modification; Destabilizing modifications of the CH3 domain of the first polypeptide do not destabilize the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; destabilizing modifications of the CH3 domain of the second polypeptide do not destabilize the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The first polypeptide or the second polypeptide further comprises a payload portion, providing a polypeptide complex.

[0017] In some embodiments, the first polypeptide and / or the second polypeptide further comprises a functional moiety.

[0018] In some embodiments, destabilization modification of the CH3 domain of the first polypeptide stabilizes the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; and / or destabilization modification of the CH3 domain of the second polypeptide stabilizes the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide.

[0019] In some embodiments, the first polypeptide includes a payload portion and the second polypeptide includes a functional portion, or the first polypeptide includes a functional portion and the second polypeptide includes a payload portion.

[0020] In some embodiments, (a) The CH3 domain of the first polypeptide contains 366W and 370E; the CH3 domain of the second polypeptide contains 407V, 366S, 368A and 357K; or (b) The CH3 domain of the first polypeptide contains 366W, 370E and 354C; the CH3 domain of the second polypeptide contains 407V, 366S, 368A, 357K and 349C; or (c) The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 24; the CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 25; or (d) The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 26; the CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 27.

[0021] In some embodiments, the payload portion is or includes a detectable portion, a fluorescent portion, a luminescent portion, a radiopaque / contrast agent, a radiolabel, an immunodetectable portion, a portion having detectable activity, an enzyme portion, a drug portion, or a cytotoxic portion.

[0022] In some embodiments, the functional portion is or comprises a binding portion, an antibody or its target-binding fragment or derivative, a target-binding peptide / polypeptide, a target-binding nucleic acid, a detectable portion, a fluorescent portion, a luminescent portion, a radiopaque / contrast agent, a radiolabel, an immunodetectable portion, a portion having detectable activity, an enzyme portion, a drug portion, or a cytotoxic portion.

[0023] In some embodiments, the first polypeptide and / or the second polypeptide further comprises a CH2 domain and / or a hinge region.

[0024] This disclosure also relates to a polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide contains a knob modification, and the CH3 domain of the second polypeptide contains a hole modification; The CH3 domain of the first polypeptide and / or the CH3 domain of the second polypeptide include destabilizing modifications to destabilize the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The first polypeptide and / or the second polypeptide further comprises a payload portion, providing a polypeptide complex.

[0025] In some embodiments, (a) the CH3 domain of the first polypeptide contains 370E and the CH3 domain of the second polypeptide contains 357E; or (b) The CH3 domain of the first polypeptide contains 370K, and the CH3 domain of the second polypeptide contains 357K.

[0026] In some embodiments, (a) The CH3 domain of the first polypeptide contains 366W and 370E; the CH3 domain of the second polypeptide contains 407V, 366S, and 368A; or (b) The CH3 domain of the first polypeptide contains 366W; the CH3 domain of the second polypeptide contains 407V, 366S, 368A and 357K; or (c) The CH3 domain of the first polypeptide contains 366W, 370E and 354C; the CH3 domain of the second polypeptide contains 407V, 366S and 368A; or (d) The CH3 domain of the first polypeptide contains 366W; the CH3 domain of the second polypeptide contains 407V, 366S, 368A, 357K and 349C; or (e) The CH3 domain of the first polypeptide contains 366W and 370E; the CH3 domain of the second polypeptide contains 407V, 366S, 368A and 349C; or (f) The CH3 domain of the first polypeptide contains 366W and 354C; the CH3 domain of the second polypeptide contains 407V, 366S, 368A, and 357K; or (g) The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 48; the CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 25; or (h) The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 26; the CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 49; or (i) The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 48; the CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 27; or (j) The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 24; the CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 51; or (k) The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 50; the CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 25.

[0027] In some embodiments, the payload portion is or includes a detectable portion, a fluorescent portion, a luminescent portion, a radiopaque / contrast agent, a radiolabel, an immunodetectable portion, a portion having detectable activity, an enzyme portion, a drug portion, or a cytotoxic portion.

[0028] In some embodiments, the first polypeptide and / or the second polypeptide further comprises a CH2 domain and / or a hinge region.

[0029] explanation This disclosure relates to a variation of ForCE technology that can be used in the production of payload-carrying molecules (such as ADCs) by combining functional (e.g., binding) entities with payload-coupling Fc molecules. This opens up a robust, rapid, and reliable pathway for generating a defined matrix of ADCs, in which different binders of various formats are attached to payloads such as small molecules, peptides, nucleic acids, and proteins via various linkers in a defined position and stoichiometry.

[0030] The principle on which this disclosure is based is shown in the schematic diagram in Figure 1.

[0031] The first “donor” precursor polypeptide complex comprises a first polypeptide and a second polypeptide, each containing a CH3 domain. The CH3 domains of the first and second polypeptides contain complementary modifications to facilitate their association into the polypeptide complex (in the example in Figure 1, a “knob-into-hole” modification). The CH3 domains of the first and second polypeptides of the “donor” precursor polypeptide complex further contain one or more destabilizing modifications to destabilize the interaction between the first and second polypeptides. One or both polypeptides further contain a payload portion.

[0032] The second “acceptor” precursor polypeptide complex similarly contains a first polypeptide and a second polypeptide, each containing a CH3 domain. The CH3 domains of the first and second polypeptides include complementary modifications to facilitate their association into the polypeptide complex (a “knob-into-hole” modification in the example in Figure 1). The CH3 domains of the first and second polypeptides in the “acceptor” precursor polypeptide complex further include one or more destabilizing modifications to destabilize the interaction between the first and second polypeptides. One or both polypeptides may further contain a functional moiety (the Fab fragment in the example in Figure 1).

[0033] The constituent polypeptides of the "donor" precursor complex associate with the polypeptides of the "acceptor" precursor complex at a higher affinity than the affinity with which they associate with their interaction partners in the "donor" precursor complex. Similarly, the constituent polypeptides of the "acceptor" precursor complex associate with the polypeptides of the "donor" precursor complex at a higher affinity than the affinity with which they associate with their interaction partners in the "acceptor" precursor complex. This is achieved by destabilizing modifications of the polypeptides of the donor and acceptor precursor complexes. Destabilizing modifications of the polypeptides of the "acceptor" precursor polypeptide complex do not destabilize the association between the polypeptides of the "acceptor" precursor polypeptide complex and the polypeptides of the "donor" precursor polypeptide complex. Similarly, destabilizing modifications of the polypeptides of the "donor" precursor polypeptide complex do not destabilize the association between the polypeptides of the "donor" precursor polypeptide complex and the polypeptides of the "acceptor" precursor polypeptide complex.

[0034] Destabilizing modifications of the polypeptide in the "acceptor" precursor polypeptide complex can stabilize the association between the polypeptide of the "acceptor" precursor polypeptide complex and the polypeptide of the "donor" precursor polypeptide complex. Similarly, destabilizing modifications of the polypeptide in the "donor" precursor polypeptide complex can stabilize the association between the polypeptide of the "donor" precursor polypeptide complex and the polypeptide of the "acceptor" precursor polypeptide complex. For example, as shown in Figure 1, destabilizing modifications of the CH3 domain of the polypeptide in the "donor" precursor complex may introduce an amino acid residue that has a repulsive charge to the charge of an interacting amino acid residue in the other polypeptides of the "donor" precursor complex, and this same amino acid residue may contribute to the formation of a salt crosslink with an amino acid residue in the CH3 domain of the polypeptide of the "acceptor" precursor complex. Similarly, destabilizing modifications of the CH3 domain of the polypeptide in the "acceptor" precursor complex may introduce an amino acid residue that has a repulsive charge to the charge of an interacting amino acid residue in the other polypeptides of the "acceptor" precursor complex, and this same amino acid residue may contribute to the formation of a salt crosslink with an amino acid residue in the CH3 domain of the polypeptide of the "donor" precursor complex.

[0035] Modifications to facilitate the association between constituent polypeptides of the "donor" precursor complex and modifications to facilitate the association between constituent polypeptides of the "acceptor" precursor complex are preferably suited to facilitating the association between polypeptides of the "donor" precursor complex and polypeptides of the "acceptor" precursor complex. For example, as shown in Figure 1, the CH3 domain of the polypeptide of the "donor" precursor complex may include a knob modification to facilitate association with the CH3 domain of the polypeptide of the "acceptor" precursor complex, which may include a hole modification. Similarly, the CH3 domain of the polypeptide of the "donor" precursor complex may include a hole modification to facilitate association with the CH3 domain of the polypeptide of the "acceptor" precursor complex, which may include a knob modification.

[0036] When "donor" and "acceptor" precursor complexes are incubated with each other, polypeptide exchange between the "donor" and "acceptor" complexes results in the formation of two new complexes: (i) a final payload-supported complex (in the example in Figure 1, "defined labeled antibody") containing the payload-supported polypeptide of the "donor complex" and optionally the polypeptide of the "acceptor complex" supporting the functional portion; and (ii) a by-product "dummy" complex (in the example in Figure 1, "dummy dimer") containing polypeptides of the "donor" and "acceptor" precursor complexes that are not included in the final payload-supported complex.

[0037] Polypeptide complex Aspects of this disclosure relate to polypeptide complexes. In this specification, “polypeptide complex” refers to a complex formed by protein-protein interactions between two or more polypeptide monomers. In this specification, “polypeptide” refers to a polymer chain of multiple amino acid monomers linked by peptide bonds.

[0038] Polypeptide complexes can be formed by non-covalent and / or covalent interactions between their constituent polypeptides. Non-covalent interactions include, for example, electrostatic interactions (e.g., ionic bonds, salt bridges), hydrogen bonds, van der Waals forces, and hydrophobic interactions. Covalent interactions include, for example, disulfide bonds. It will be understood that the interactions forming polypeptide complexes involve amino acid / amino acid sequences derived from different polypeptide monomers (i.e., interchain interactions).

[0039] In aspects and embodiments of this disclosure, the constituent polypeptides of the polypeptide complex include CH3 domains, and the polypeptide complex is formed by interactions including association between the CH3 domains of the constituent polypeptides of the polypeptide complex. In some embodiments, the polypeptide complex is formed by interactions including disulfide bonds between the CH3 regions of the constituent polypeptides of the polypeptide complex.

[0040] In aspects and embodiments of this disclosure, the constituent polypeptides of the polypeptide complex include CH2 domains, and the polypeptide complex is formed by interactions including association between the CH2 domains of the constituent polypeptides of the polypeptide complex.

[0041] In aspects and embodiments of this disclosure, the constituent polypeptides of the polypeptide complex include hinge regions, and the polypeptide complex is formed by interactions including association between the hinge regions of the constituent polypeptides of the polypeptide complex. In some embodiments, the polypeptide complex is formed by interactions including disulfide bonds between the hinge regions of the constituent polypeptides of the polypeptide complex.

[0042] In some embodiments, the constituent polypeptides of the polypeptide complex according to the Disclosure include CH3 domains and CH2 domains. In such embodiments, the constituent polypeptides of the polypeptide complex may interact with each other through association between their CH3 and / or CH2 regions to form an Fc region. That is, in some embodiments, the polypeptide complex according to the Disclosure may be an Fc region or may include an Fc region.

[0043] As used herein, “Fc region” refers to a polypeptide complex formed by the interaction of polypeptides, each containing a CH2 domain and a CH3 domain. In preferred embodiments, the Fc region may be a polypeptide complex formed by the interaction of polypeptides, each containing the following structure: N-terminus-[...]-[CH2 domain]-[CH3 domain]-[...]-C-terminus.

[0044] When used in the representation of polypeptide structures herein, "[...]" indicates the presence of any additional protein domains / regions. For example, in the structure in the last sentence of the previous paragraph, additional protein domains / regions are optionally located downstream of the CH3 domain, prior to the C-terminus of the polypeptide. Furthermore, when used in the representation of polypeptide structures herein, "-" indicates an optional linker sequence. For example, in the structure in the last sentence of the previous paragraph, a linker sequence may be optionally provided between the CH2 domain and the CH3 domain.

[0045] The Fc region provides interactions with Fc receptors and other molecules of the immune system to produce functional effects. IgG Fc-mediated effector functions are outlined, for example, Jefferis et al., Immunol Rev 1998 163:59-76 (the entire text of which is incorporated herein by reference), and are mediated by Fc-mediated recruitment and activation of immune cells (e.g., macrophages, dendritic cells, neutrophils, basophils, eosinophils, platelets, mast cells, NK cells, and T cells) via interactions between the Fc region and Fc receptors expressed by immune cells, recruitment of complement pathway components via binding of the Fc region to the complement protein C1q, and consequently activation of the complement cascade. Fc-mediated functions include Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cell-mediated cytotoxicity (CDC), membrane attack complex (MAC) formation, cell degranulation, cytokine and / or chemokine production, and antigen processing and presentation.

[0046] The polypeptide complexes described herein are broadly classified into four classes: “donor” precursor complexes, “acceptor” precursor complexes, final payload-supporting complexes, and by-product “dummy” complexes.

[0047] The precursor complex is formed by interactions between polypeptides containing CH3 domains, and this interaction involves the association of the CH3 domains of the polypeptides. The CH3 domains of the polypeptides include modifications to facilitate their association, as described below, for example. In particular, the CH3 domains of the polypeptides in the precursor complex may include paired “knob” and “hole” modifications, as described below. Importantly, the polypeptides in the precursor complex also include modifications to one or both of the CH3 domains to destabilize the association between the CH3 domains. The destabilization of the association borne by the destabilizing modification should not be so great as to substantially prevent the interaction between polypeptides, and therefore the formation of the precursor complex.

[0048] One or both polypeptides of the "donor" precursor polypeptide complex further comprise a payload portion. One or both polypeptides of the "acceptor" precursor polypeptide complex may further comprise a functional portion.

[0049] The constituent polypeptides of the "donor" precursor complex associate with the polypeptides of the "acceptor" precursor complex at a higher affinity than the affinity with which they associate with their interaction partners in the "donor" precursor complex. Similarly, the constituent polypeptides of the "acceptor" precursor complex associate with the polypeptides of the "donor" precursor complex at a higher affinity than the affinity with which they associate with their interaction partners in the "acceptor" precursor complex. This is achieved by destabilizing modifications of the polypeptides of the donor and acceptor precursor complexes. Destabilizing modifications of the polypeptides of the "acceptor" precursor complex do not destabilize the association between the polypeptides of the "acceptor" precursor complex and the polypeptides of the "donor" precursor complex. Similarly, destabilizing modifications of the polypeptides of the "donor" precursor complex do not destabilize the association between the polypeptides of the "donor" precursor complex and the polypeptides of the "acceptor" precursor complex. Modifications to promote the association between the constituent polypeptides of the "donor" precursor complex and modifications to promote the association between the constituent polypeptides of the "acceptor" precursor complex are preferably suited to promoting the association between the polypeptides of the "donor" precursor complex and the polypeptides of the "acceptor" precursor complex.

[0050] The final payload-supported complex is formed by interactions between polypeptides containing CH3 domains, and these interactions include association between the CH3 domains of the polypeptides. The polypeptides of the complex further include the payload portion. The CH3 domains of the polypeptides include modifications to facilitate their association, as described below, for example. In particular, the CH3 domains of the polypeptides of the precursor complex may include paired “knob” and “hole” modifications, as described below. The association between the constituent polypeptides of the final payload-supported complex is stronger than the association between the constituent polypeptides of the precursor complex described above. That is, the final payload-supported complex has greater stability (e.g., a lower tendency to dissociate) compared to the precursor complex. In some embodiments, the polypeptides of the final payload-supported complex include modifications to one or both of the CH3 domains to stabilize the association between the CH3 domains. In some embodiments, modifications that are destabilizing modifications of the constituent polypeptides of the precursor complex are modifications that stabilize the association between the CH3 domains of the polypeptides of the final payload-supported complex.

[0051] The byproduct "dummy" complex is similarly formed by interactions between polypeptides containing CH3 domains, and these interactions include association between the CH3 domains of the polypeptides. The CH3 domains of the polypeptides include modifications to facilitate their association, as described below, for example. In particular, the CH3 domains of the polypeptides of the precursor complex may include paired "knob" and "hole" modifications, as described below. The association between the constituent polypeptides of the byproduct "dummy" complex is stronger than the association between the constituent polypeptides of the precursor complex described above. That is, the byproduct "dummy" complex has greater stability compared to the precursor complex (e.g., less tendency to dissociate). In some embodiments, the polypeptides of the byproduct "dummy" complex include modifications to one or both of the CH3 domains to stabilize the association between the CH3 domains. In some embodiments, modifications that are destabilizing modifications of the constituent polypeptides of the precursor complex are modifications that stabilize the association between the CH3 domains of the polypeptides of the byproduct "dummy" complex.

[0052] It will be understood that the final payload-supported complex according to this disclosure is more stable / less prone to dissociation / formation by polypeptides that interact with it at a greater affinity compared to the precursor polypeptide complex according to this disclosure (e.g., the “donor” or “acceptor” polypeptide complex).

[0053] Similarly, the by-product “dummy” complexes of the present disclosure are more stable / less prone to dissociation / formation by polypeptides that interact with them at a greater affinity compared to the precursor polypeptide complexes of the present disclosure (e.g., the “donor” or “acceptor” polypeptide complexes).

[0054] CH3 domain In this specification, the "CH3 domain" refers to the amino acid sequence corresponding to the CH3 domain of immunoglobulin (Ig). The CH3 domain is the region of Ig formed by positions 341-447 of the immunoglobulin constant domain according to the EU numbering system described in Edelman et al., Proc. Natl. Acad. Sci. USA (1969) 63(1):78-85.

[0055] In some embodiments, the CH3 domain corresponds to or is derived from the CH3 domain of Ig derived from mammals (e.g., eutherians, placental mammals, epithelioid mammals, prothorians, ancient hominids, primates (rhesus monkeys, crab-eating macaques, non-human primates, or humans)). In some embodiments, the CH3 domain corresponds to or is derived from the CH3 domain of Ig derived from humans.

[0056] In some embodiments, the CH3 domain corresponds to or is derived from the CH3 domain of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM.

[0057] In some embodiments, the CH3 domain corresponds to or is derived from the CH3 domain of human IgG (e.g., hIgG1, hIgG2, hIgG3, hIgG4), hIgA (e.g., hIgA1, hIgA2), hIgD, hIgE, or hIgM. In some embodiments, the CH3 domain corresponds to or is derived from the CH3 domain of human IgG1 allotype (e.g., G1m1, G1m2, G1m3, or G1m17).

[0058] The CH3 domain of the human IgG1 G1m1 allotype is formed at positions 224-330 of UniProt P01857-1,v1 and has the amino acid sequence shown in SEQ ID NO: 1. The CH3 domain of the human IgG1 G1m3 allotype is shown in SEQ ID NO: 2. The CH3 domain of human IgG2 is formed at positions 220-326 of UniProt P01859-1,v2 and has the amino acid sequence shown in SEQ ID NO: 3. The CH3 domain of human IgG3 is formed at positions 271-376 of UniProt P01860-1,v2 and has the amino acid sequence shown in SEQ ID NO: 4. The CH3 domain of human IgG4 is formed at positions 221-327 of UniProt P01861-1,v1 and has the amino acid sequence shown in SEQ ID NO: 5. The third Ig-like region of human IgA1 is formed at positions 228-330 of UniProt P01876-1,v2 and has the amino acid sequence shown in SEQ ID NO: 6. The third Ig-like region of human IgA2 is formed at positions 215-317 of UniProt P01877-1,v4 and has the amino acid sequence shown in SEQ ID NO: 7. The third Ig-like region of human IgD is formed at positions 267-373 of UniProt P01880-1,v3 and has the amino acid sequence shown in SEQ ID NO: 8. The third Ig-like region of human IgE is formed at positions 214-318 of UniProt P01854-1,v1 and has the amino acid sequence shown in SEQ ID NO: 9. The CH3 domain of human IgM is formed at positions 218-323 of UniProt P01871-1,v4 and has the amino acid sequence shown in SEQ ID NO: 10.

[0059] In this specification, an amino acid sequence that "corresponds" to a specified domain / region of a reference polypeptide, or a reference amino acid sequence, has at least 60% amino acid sequence identity with the amino acid sequence of the reference domain / region or the reference amino acid sequence, for example, at least ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%. An amino acid sequence that "corresponds" to a specific domain / region of a reference polypeptide or a reference amino acid sequence can be identified by sequence alignment of the target sequence to the reference sequence using sequence alignment software such as ClustalOmega (Soding, J.2005, Bioinformatics 21, 951-960).

[0060] The amino acid sequence "derived" from a specified domain / region of the reference polypeptide, or the reference amino acid sequence, has at least 60% amino acid sequence identity with the amino acid sequence of the reference domain / region or the reference amino acid sequence, for example, at least ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%. In some embodiments, the amino acid sequence derived from a specific domain / region of the reference polypeptide, or the reference amino acid sequence, has at least 60% of one of the amino acid sequences, for example, at least ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99%, compared to the amino acid sequence of the reference domain / region or the reference amino acid sequence, and is not identical to the amino acid sequence of the reference domain / region or the reference amino acid sequence. In some embodiments, the amino acid sequence derived from a specific domain / region of the reference polypeptide, or the reference amino acid sequence, includes one or more (e.g., 1, 2, 3, 4, 5, 7, 8, 10 or more) differences compared to the amino acid sequence of the reference domain / region or the reference amino acid sequence.

[0061] As an example, the CH3 domain of human IgG2 is formed at positions 220-326 of UniProt P01859-1,v2 and has the amino acid sequence shown in Sequence ID No. 3. It will be understood that positions 220-326 of UniProt P01859-1,v2 correspond to positions 224-330 of UniProt P01857-1,v1.

[0062] In some embodiments, the CH3 domain according to the present disclosure comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, for example, one of the following amino acid sequence identity values: ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%.

[0063] In some embodiments, the CH3 domain includes or comprises an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NOs: 1, 2, 3, 4, or 5, for example, one of the following amino acid sequence identity levels: ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%.

[0064] In some embodiments, the CH3 domain includes or comprises an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO: 1, for example, one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity.

[0065] In some embodiments, the CH3 domain includes or consists of an amino acid sequence having at least 70% amino acid sequence identity with one of sequence numbers 24-143, for example, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity.

[0066] In some embodiments, the CH3 domain includes or consists of an amino acid sequence having at least 70% amino acid sequence identity with one of sequence numbers 24-119, for example, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity.

[0067] As described below, in various aspects and embodiments of this disclosure, the CH3 domain includes one or more modifications, such as modifications that affect the association between CH3 domains. If the CH3 domain described herein includes modifications and further includes or consists of amino acid sequences within a certain threshold percentage amino acid sequence identity to a reference amino acid sequence, it will be understood that any mutation to the reference amino acid sequence is limited to a reference sequence location other than the modified location. For example, in the example of a CH3 domain including a destabilizing modification and containing an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 1, the CH3 domain necessarily includes a destabilizing modification, so that up to 30% of mutations from the amino acid sequence of SEQ ID NO: 1 are limited to locations other than the modified location. As a further example, in the example of a CH3 domain including 366W and 370E and containing an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 24, up to 30% of mutations from the amino acid sequence of SEQ ID NO: 24 are limited to locations of the CH3 domain sequence other than positions 366 and 370, which are necessarily 366W and 370E.

[0068] Modifications to CH3 domains that affect the association between CH3 domains The CH3 domains described herein may include modifications to facilitate association with other CH3 domains, and may also include modifications to destabilize association with specific CH3 domains.

[0069] In some embodiments and aspects, the CH3 domains of the Disclosure include modifications to facilitate association with other CH3 domains. In some embodiments, the CH3 domains include modifications to facilitate heteromerization, i.e., association between non-identical CH3 domains.

[0070] Modifications to CH3 domains to facilitate association with other CH3 domains are known in the art and include paired modifications to CH3 regions (associating with each other) described in Ha et al., Front.Immunol (2016) 7:394 (which is incorporated herein by reference in its entirety), particularly those listed in Table 1. Such modifications include modifications to KiH, KiHs-s, HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, SEED, and A107 techniques.

[0071] In aspects and embodiments of this disclosure, the interactions between constituent polypeptides of the polypeptide complex described herein are enhanced by “knob-into-hole” technology. Knob-into-hole (or “KiH”) technology is described, for example, in International Publication No. 96 / 027011, Ridgway, JB, et al., Protein Eng. 9 (1996) 617-621, Merchant, AM, et al., Nat. Biotechnol. 16 (1998) 677-681, U.S. Patent No. 7,695,936, and Carter, J. Immunol. Meth. (2001) 248, 7-15, all of which are incorporated herein by reference in their entirety.

[0072] Heterodimerization between CH3 domain-supported polypeptides is facilitated by modifications to their interaction surfaces to provide “knob” and “hole” modifications complementary to the amino acid sequence of the polypeptide’s CH3 domain. The “knob” and “hole” modifications are positioned within each CH3 domain so that the “knob” can be placed in the “hole” to promote heterodimerization (and inhibit homodimerization) and / or stabilize the heterodimer. Knobs are constructed by substituting amino acids with smaller side chains with amino acids with larger side chains (e.g., tyrosine or tryptophan). Holes are created by substituting amino acids with larger side chains with amino acids with smaller side chains (e.g., valine, alanine, serine, or threonine).

[0073] For example, additional interchain disulfide crosslinks between CH3 domains can be introduced by introducing a cysteine ​​residue at position 354 of the CH3 domain having a "knob" modification, and by introducing a cysteine ​​residue at position 349 of the CH3 domain having a "hole" modification (as described in Merchant, AM, et al., Nature Biotech. 16(1998) 677-681). A knob modification that further includes the introduction of a cysteine ​​residue to form interchain disulfide crosslinks may be called a "knob-cys" modification, and similarly, a hole modification that further includes the introduction of a cysteine ​​residue to form interchain disulfide crosslinks may be called a "hole-cys" modification.

[0074] As used herein, “modification” refers to a difference from a reference amino acid sequence. The reference amino acid sequence may be the amino acid sequence encoded by the most common nucleotide sequence of the gene encoding the relevant protein.

[0075] As an example, in embodiments of this specification, a particular CH3 domain may include a knob modification that includes a modification at position 366 of the CH3 domain (unless otherwise specified, the numbering of positions or substitutions of CH3 regions herein follows the EU numbering system described in Edelman et al., Proc. Natl. Acad. Sci. USA (1969) 63(1):78-85). The modification may provide a tryptophan residue at position 366, which is a threonine residue in the standard sequence of human IgG1 / IgG2 / IgG3 / IgG4.

[0076] In the embodiments of this specification (and more generally in the art), “modification” may also be called “substitution” or “mutation.”

[0077] Modifications typically involve the substitution of amino acid residues with non-identical “replacement” amino acid residues. The replacement amino acid residues in modifications according to this disclosure may be native amino acid residues (i.e., those encoded by the genetic code) selected from alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile):leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val), which are not identical to the amino acid residues at the relevant positions in the pre-modification amino acid sequence. In some embodiments, the replacement amino acid residues in modifications may be amino acid residues that do not exist naturally, i.e., amino acid residues other than those listed in the preceding sentence. Examples of non-natural amino acid residues include norleucine, ornithine, norvaline, homoserine, aib, and other amino acid residue analogs, such as those described in Ellman, et al., Meth. Enzym. 202 (1991) 301-336.

[0078] The amino acids used are, optionally, in their L-forms. The terms "positively charged" or "negatively charged" amino acids refer to the amino acid side-chain charge at pH 7.4. Amino acids can be classified as follows according to their common side-chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile, Trp, Tyr, Phe; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic or negatively charged Asp, Glu; (4) Basic or positively charged: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro. [table] TIFF2026509081000002.tif149170

[0079] In some embodiments, the hydrophobic amino acid is selected from norleucine, Met, Ala, Val, Leu, Ile, Trp, Tyr, and Phe. In some embodiments, the hydrophobic amino acid is selected from Ala, Val, Leu, Ile, and Tyr. In some embodiments, the hydrophobic amino acid is Val, Leu, or Ile. In some embodiments, the hydrophobic amino acid is Leu or Ile. In some embodiments, the hydrophobic amino acid is Leu. In some embodiments, the hydrophobic amino acid is Tyr. In some embodiments, the hydrophobic amino acid is Phe.

[0080] In some embodiments, the positively charged amino acid is His, Lys, or Arg. In some embodiments, the positively charged amino acid is Lys or Arg. In some embodiments, the positively charged amino acid is Lys.

[0081] In some embodiments, the negatively charged amino acid is Asp or Glu.

[0082] In embodiments and aspects of this disclosure, the CH3 domain—for example, the CH3 domain of the constituent polypeptide of the polypeptide complex according to this disclosure—is a pair of CH3 domains "KiH" or "KiH" S-S This includes modifications.

[0083] In some embodiments, the CH3 domain including the knob modification contains a tryptophan or tyrosine residue (i.e., 366W or 366Y) at position 366. In some embodiments, the knob modification is T366W or T366Y, or includes them.

[0084] In some embodiments, the CH3 domain including the knob modification includes 366W. In some embodiments, the knob modification is or includes T366W.

[0085] In this specification, when referring to the location of the immunoglobulin constant region (e.g., following Eu numbering), the corresponding location in the homologous sequence to the constant region sequence of human IgG1 (G1m1 allotype) is also intended. The location corresponding to that identified in the CH3 domain of human IgG1 (G1m1 allotype) can be identified by sequence alignment, which can be performed using sequence alignment software such as ClustalOmega (Soding, J.2005, Bioinformatics 21, 951-960).

[0086] For example, in human IgG1, 366T (position 26 of SEQ ID NO: 1) corresponds to position 26 of SEQ ID NO: 2 (hIgG1 G1m3 allotype), position 26 of SEQ ID NO: 3 (hIgG2), position 26 of SEQ ID NO: 4 (hIgG3), and position 26 of SEQ ID NO: 5 (hIgG4).

[0087] Furthermore, when a modification is indicated herein by reference to a reference amino acid in the constant region of human IgG1 (G1m1 allotype), it will be understood that when such a modification is provided in its homologous protein / domain, the intended substitution is at an equivalent position in its sequence. Of importance are the amino acid residues at the relevant positions after modification. For example, human IgG1 (G1m1) contains D at position 356, and human IgG1 (G1m3) contains E at position 356. The "CH3 domain containing D356K" of course includes the CH3 domain having the sequence of SEQ ID NO: 1 and containing the amino acid substitution D356K, but also includes, for example, the CH3 domain having the sequence of SEQ ID NO: 2 and containing the amino acid substitution E356K.

[0088] In some embodiments, the CH3 domains including the Hall modification include 407V, 407A, 407S or 407T; 366S, 366V or 366A; and 368A, 368V, 368S or 368T. In some embodiments, the Hall modification is Y407V, Y407A, Y407S or Y407T; T366S, T366V or T366A; and L368A, L368V, L368S or L368T, or includes them.

[0089] In some embodiments, the CH3 domain including the Hall modification includes 407V, 366S, and 368A. In some embodiments, the Hall modification is Y407V, T366S, and L368A, or includes them.

[0090] In aspects and embodiments of the present disclosure, the CH3 domains of the polypeptides in the polypeptide complex of the present disclosure include modifications for the formation of interchain disulfide bonds (i.e., between polypeptides). Such modifications may include the introduction of one or more cysteine ​​residues into one or both of the CH3 domains of the constituent polypeptides of the polypeptide complex of the present disclosure. More specifically, such modifications may result in the CH3:CH3 interface formed between the CH3 domains of the polypeptides in the polypeptide complex of the present disclosure including disulfide bonds formed between cysteine ​​residues (derived from each polypeptide).

[0091] In some embodiments, the polypeptide complex according to the present disclosure (e.g., the final payload-carrying polypeptide complex) comprises (i) a polypeptide having a CH3 domain comprising one modification from row 1 to 6 of column A in Table I; and (ii) a polypeptide having a CH3 domain comprising one modification from row 1 to 6 of column B in Table I. [table] TIFF2026509081000003.tif53170

[0092] In preferred embodiments, for a given polypeptide complex (e.g., the final payload-supporting polypeptide complex), modifications of the CH3 domain, including modifications from column A, and modifications of the CH3 domain, including modifications from column B, are selected from the same row in Table I. For example, in some embodiments, the polypeptide complex according to the present disclosure comprises (i) a polypeptide having a CH3 domain containing Y349C (row 2 of column A); and (ii) a polypeptide having a CH3 domain containing S354C (row 2 of column B).

[0093] In some embodiments, the polypeptides according to the Disclosure (e.g., precursor polypeptide complexes) include modifications that introduce one or more cysteine ​​residues into only one of the CH3 domains of the polypeptide complex. Such modifications may introduce cysteine ​​residues that do not participate in the formation of disulfide bonds in the polypeptide complex. Such modifications may introduce cysteine ​​residues that do not participate in the formation of interchain disulfide bonds in the precursor polypeptide complex but do participate in the formation of interchain disulfide bonds in the final payload-carrying polypeptide complex of the Disclosure.

[0094] As an example, the final payload-carrying polypeptide complex (C) of the present disclosure may be formed by polypeptide chain exchange between a first precursor polypeptide complex (A) and a second precursor polypeptide complex (B); (A) comprises (i) a first polypeptide comprising 349C and (ii) a second polypeptide comprising 354S; (B) comprises (i) a first polypeptide comprising 349Y and (ii) a second polypeptide comprising 354C; and (C) comprises the first polypeptide of (A) (i.e., comprising 349C) and the second polypeptide of (B) (i.e., comprising 354C).

[0095] Accordingly, in some embodiments, the polypeptide complex according to the present disclosure (e.g., a precursor polypeptide complex) is (i) a polypeptide having a CH3 domain including one modification from rows 1 to 6 of column A in Table I; and (ii) a polypeptide having a CH3 domain lacking one modification from rows 1 to 6 of column B in Table I; or (i) a polypeptide having a CH3 domain lacking one modification from rows 1 to 6 of column A in Table I; and (ii) a polypeptide having a CH3 domain including one modification from rows 1 to 6 of column B in Table I (in preferred embodiments, for a given polypeptide complex, the modifications of the CH3 domain from column A and the modifications of the CH3 domain from column B are selected from the same row in Table I).

[0096] In some embodiments, the CH3 domain including the knob modification further includes 354C. In some embodiments, the CH3 domain including the knob modification further includes modified S354C.

[0097] In some embodiments, the CH3 domain including the knob modification includes 366W or 366Y and 354C. In some embodiments, the knob modification is T366W or T366Y and S354C, or includes them.

[0098] In some embodiments, the CH3 domain including the knob modification includes 366W and 354C. In some embodiments, the knob modification is T366W and S354C, or includes them.

[0099] In some embodiments, the CH3 domain including the hole modification further includes 349C. In some embodiments, the CH3 domain including the hole modification further includes modified Y349C.

[0100] In some embodiments, the CH3 domains including Hall modifications include 407V, 407A, 407S or 407T; 366S, 366V or 366A; 368A, 368V, 368S or 368T; and 349C. In some embodiments, the Hall modifications are Y407V, Y407A, Y407S or Y407T; T366S, T366V or T366A; L368A, L368V, L368S or L368T; and Y349C, or include them.

[0101] In some embodiments, the CH3 domain including the Hall modification includes 407V, 366S, 368A, and 354C. In some embodiments, the Hall modification is Y407V, T366S, L368A, and Y354C, or includes them.

[0102] In some embodiments, the polypeptide complex according to the present disclosure comprises (i) a first polypeptide having a CH3 domain including a knob modification, and (ii) a second polypeptide having a CH3 domain including a hole modification.

[0103] In some embodiments, the polypeptide complex according to the present disclosure comprises (i) a first polypeptide having a CH3 domain including 366W, and (ii) a second polypeptide having a CH3 domain including 407V, 366S, and 368A.

[0104] In some embodiments, the polypeptide complex according to the present disclosure (e.g., the final payload-supported polypeptide complex) comprises (i) a first polypeptide having CH3 domains including 366W and 354C, and (ii) a second polypeptide having CH3 domains including 407V, 366S, 368A and 349C.

[0105] In some embodiments, the polypeptide complex according to the present disclosure (e.g., a precursor polypeptide complex) comprises (i) a first polypeptide having CH3 domains including 366W and 354C, and (ii) a second polypeptide having CH3 domains including 407V, 366S, 368A and 349Y.

[0106] In some embodiments, the polypeptide complex according to the present disclosure (e.g., a precursor polypeptide complex) comprises (i) a first polypeptide having CH3 domains including 366W and 354S, and (ii) a second polypeptide having CH3 domains including 407V, 366S, 368A and 349C.

[0107] In embodiments and aspects of the present disclosure, the CH3 domain—for example, the CH3 domain of a constituent polypeptide of the polypeptide complex according to the present disclosure—includes, for example, the paired CH3 domain "DD-KK" modification described in U.S. Patent No. 8,592,562. In some embodiments, the CH3 domain includes 392D and 409D. In some embodiments, the CH3 domain includes 356K and 399K. In some embodiments, the polypeptide complex according to the present disclosure includes (i) a first polypeptide having CH3 domains including 392D and 409D, and (ii) a second polypeptide having CH3 domains including 356K and 399K.

[0108] In embodiments and aspects of the present disclosure, the CH3 domain—for example, the CH3 domain of a constituent polypeptide of the polypeptide complex according to the present disclosure—includes, for example, the paired CH3 domain "Duobody" modification described in Labrijn et al., Proc. Natl. Acad. Sci. US A. (2013) 110(13): 5145-50. In some embodiments, the CH3 domain includes 409R. In some embodiments, the CH3 domain includes 405L. In some embodiments, the polypeptide complex according to the present disclosure includes (i) a first polypeptide having a CH3 domain including 409R, and (ii) a second polypeptide having a CH3 domain including 405L.

[0109] In embodiments and aspects of the present disclosure, the CH3 domain—for example, the CH3 domain of a constituent polypeptide of the polypeptide complex according to the present disclosure—includes, for example, the paired CH3 domain "EEE-RRR" modification described in Strop et al., J.Mol.Biol.(2012)420(3):204-19. In some embodiments, the CH3 domain includes 368E. In some embodiments, the CH3 domain includes 409R. In some embodiments, the polypeptide complex according to the present disclosure includes (i) a first polypeptide having a CH3 domain including 368E, and (ii) a second polypeptide having a CH3 domain including 409R.

[0110] In embodiments and aspects of the present disclosure, the CH3 domain—for example, the CH3 domain of the constituent polypeptide of the polypeptide complex according to the present disclosure—includes, for example, the paired CH3 domain "EW-RVT" modification described in Choi et al., Mol. Cancer Ther. (2013) 12(12):2748-59. In some embodiments, the CH3 domain includes 360E and 409W. In some embodiments, the CH3 domain includes 347R, 399V and 405T. In some embodiments, the polypeptide complex according to the present disclosure includes (i) a first polypeptide having CH3 domains including 360E and 409W, and (ii) a second polypeptide having CH3 domains including 347R, 399V and 405T.

[0111] In embodiments and aspects of the present disclosure, the CH3 domain—for example, the CH3 domain of a constituent polypeptide of the polypeptide complex according to the present disclosure—includes the paired CH3 domain modification described in Moore et al., MAbs(2011)3(6):546-57. In some embodiments, the CH3 domain includes 364H and 405A. In some embodiments, the CH3 domain includes 349T and 394F. In some embodiments, the polypeptide complex according to the present disclosure includes (i) a first polypeptide having CH3 domains including 364H and 405A, and (ii) a second polypeptide having CH3 domains including 349T and 394F.

[0112] In embodiments and aspects of the present disclosure, the CH3 domain—for example, the CH3 domain of the constituent polypeptide of the polypeptide complex according to the present disclosure—includes the paired CH3 domain modifications described in Von Kreudenstein et al., MAbs(2013)5(5):646-54. In some embodiments, the CH3 domain includes 350V, 351Y, 405A, and 407V. In some embodiments, the CH3 domain includes 350V, 366L, 392L, and 394W. In some embodiments, the polypeptide complex according to the present disclosure includes (i) a first polypeptide having a CH3 domain including 350V, 351Y, 405A, and 407V, and (ii) a second polypeptide having a CH3 domain including 350V, 366L, 392L, and 394W.

[0113] In embodiments and aspects of the present disclosure, the CH3 domain—for example, the CH3 domain of the constituent polypeptide of the polypeptide complex according to the present disclosure—includes the paired CH3 domain modifications described in Leaver-Fay et al., Structure (2016) 24(4):641-51. In some embodiments, the CH3 domain includes 360D, 399M, and 407A. In some embodiments, the CH3 domain includes 345R, 347R, 366V, and 409V. In some embodiments, the polypeptide complex according to the present disclosure includes (i) a first polypeptide having CH3 domains including 360D, 399M, and 407A, and (ii) a second polypeptide having CH3 domains including 345R, 347R, 366V, and 409V.

[0114] In embodiments and aspects of the present disclosure, the CH3 domain—for example, the CH3 domain of the constituent polypeptide of the polypeptide complex according to the present disclosure—includes the paired CH3 domain modifications described in Choi et al., PLoS One (2015) 10(12):e0145349. In some embodiments, the CH3 domains include 370E and 409W. In some embodiments, the CH3 domains include 357N, 399V and 405T. In some embodiments, the polypeptide complex according to the present disclosure includes (i) a first polypeptide having CH3 domains including 370E and 409W, and (ii) a second polypeptide having CH3 domains including 357N, 399V and 405T.

[0115] In aspects and embodiments of this disclosure, interactions between specific CH3 domain-supported polypeptides are attenuated / inhibited / reduced by introducing one or more destabilizing modifications to the CH3 domains to destabilize the association between the CH3 domain of one CH3 domain-supported polypeptide and the CH3 domain of another CH3 domain-supported polypeptide.

[0116] As used herein, “destabilizing modification” refers to a modification that attenuates / inhibits / reduces association between a CH3 domain containing the modification and another CH3 domain. In some embodiments, a destabilizing modification is a modification to a CH3 domain that, when introduced alone, i.e., into an otherwise unmodified CH3 domain, attenuates / inhibits / reduces association between a CH3 domain containing the modification and an equivalent unmodified CH3 domain lacking the modification.

[0117] In some embodiments, the destabilizing modification is a modification to a CH3 domain that, when introduced into a first CH3 domain otherwise containing one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, attenuates / inhibits / reduces the association between the first CH3 domain and a second CH3 domain containing an equivalent amino acid sequence lacking the modification.

[0118] For example, the introduction of 357K into a CH3 domain that otherwise has the amino acid sequence of SEQ ID NO: 1 attenuates / inhibits / reduces the association between the modified CH3 domain and the CH3 domain that otherwise has the amino acid sequence of SEQ ID NO: 1. Similarly, the introduction of 370E into a CH3 domain that otherwise has the amino acid sequence of SEQ ID NO: 1 attenuates / inhibits / reduces the association between the modified CH3 domain and the CH3 domain that otherwise has the amino acid sequence of SEQ ID NO: 1.

[0119] In some embodiments, the destabilizing modification includes, or consists of, a modification to the position of CH3 provided at or near the interface when two CH3 domains interact with each other via protein-protein interactions.

[0120] In some embodiments, the destabilization modification alters the charge of amino acid residues at relevant positions in the CH3 domain compared to an equivalent CH3 domain lacking the modification. For example, the destabilization modification E357K replaces negatively charged glutamate residues with positively charged lysine residues. Similarly, the destabilization modification K370E replaces positively charged lysine residues with negatively charged glutamate residues.

[0121] In some embodiments, the destabilization modification replaces a negatively charged amino acid residue (e.g., E or D) with a positively charged amino acid residue (e.g., R, K, or H) or an uncharged amino acid residue. In some embodiments, the destabilization modification replaces a negatively charged amino acid residue with a positively charged amino acid residue.

[0122] In some embodiments, the destabilization modification replaces a positively charged amino acid residue (e.g., R, K, or H) with a negatively charged amino acid residue (e.g., E or D) or an uncharged amino acid residue. In some embodiments, the destabilization modification replaces a positively charged amino acid residue with a negatively charged amino acid residue.

[0123] In some embodiments, the destabilization modification replaces an uncharged amino acid residue with a negatively charged amino acid residue (e.g., E or D) or a positively charged amino acid residue (e.g., R, K, or H). In some embodiments, the destabilization modification replaces an uncharged amino acid residue with a negatively charged amino acid residue. In some embodiments, the destabilization modification replaces an uncharged amino acid residue with a positively charged amino acid residue.

[0124] In some embodiments, the destabilization modification disrupts one or more protein-protein interactions between amino acid residues on the first CH3 domain and amino acid residues on the second CH3 domain, which interact with the association between the first and second CH3 domains (e.g., homotype association between identical first and second CH3 domains). The protein-protein interaction may be, for example, an electrostatic interaction (e.g., a salt bridge).

[0125] As an example, the introduction of the destabilizing modification E357K into a CH3 domain otherwise having the amino acid sequence of SEQ ID NO: 1 is equivalent to introducing 357E COO into a CH3 homodimer having the amino acid sequence of SEQ ID NO: 1. - Base and 370K NH3 + It destroys the salt bridge that normally forms between the base and the substrate.

[0126] In some embodiments, the destabilization modification of the CH3 domain introduces a repulsive charge to the charge of an amino acid residue on the second CH3 domain that interacts with an equivalent, unmodified amino acid residue at the relevant position in an association between the first and second CH3 domains (e.g., a homotype association between the same first and second CH3 domains).

[0127] In some embodiments, the destabilization modification of the CH3 domain introduces an amino acid residue having the same charge as an amino acid residue on the second CH3 domain that interacts with an equivalent unmodified amino acid residue at the relevant position in an association between the first and second CH3 domains (e.g., a homotype association between the same first and second CH3 domains).

[0128] In some embodiments, the destabilization modification of the CH3 domain introduces positively charged amino acid residues, and the amino acid residues of the second CH3 domain (where equivalent unmodified amino acid residues at the relevant positions interact in the association between the first and second CH3 domains (e.g., homotype association between the same first and second CH3 domains)) are positively charged. In some embodiments, the destabilization modification of the CH3 domain introduces negatively charged amino acid residues, and the amino acid residues of the second CH3 domain (where equivalent unmodified amino acid residues at the relevant positions interact in the association between the first and second CH3 domains (e.g., homotype association between the same first and second CH3 domains)) are negatively charged.

[0129] As an example, the introduction of the destabilizing modification E357K into a CH3 domain otherwise having the amino acid sequence of SEQ ID NO: 1 would result in an interaction between a first CH3 domain containing E357K otherwise having the amino acid sequence of SEQ ID NO: 1 and a second CH3 domain having the amino acid sequence of SEQ ID NO: 1, where 357K NH3 + Base and 370K NH3 + It introduces a repulsive charge between the group and the other. Similarly, the introduction of the destabilizing modification K370E into a CH3 domain otherwise having the amino acid sequence of SEQ ID NO: 1 results in the interaction between the first CH3 domain containing K370E, which otherwise has the amino acid sequence of SEQ ID NO: 1, and the second CH3 domain having the amino acid sequence of SEQ ID NO: 1. - Base and 357E COO - A repulsive charge is introduced between the base and the element.

[0130] In some embodiments, destabilizing modifications to a CH3 domain do not destabilize the interaction between the CH3 domain and another CH3 domain containing the destabilizing modification. In some embodiments, destabilizing modifications to a CH3 domain stabilize the interaction between the CH3 domain and another CH3 domain containing the destabilizing modification.

[0131] In some embodiments and aspects, modifications to destabilize the association between a CH3 domain (CH3 domain "a") containing an destabilizing modification and a CH3 domain (CH3 domain "b") of another CH3 domain-supported polypeptide stabilize the association between CH3 domain "a" and a CH3 domain (CH3 domain "c") of another CH3 domain-supported polypeptide different from CH3 domain "b". That is, in some embodiments, modifications to CH3 domain "a" that destabilize the association between CH3 domain "a" and CH3 domain "b" stabilize the association between CH3 domain "a" and CH3 domain "c".

[0132] Similarly, in some embodiments and designs, modifications to stabilize the association between a modified CH3 domain (CH3 domain "a") and a CH3 domain of another CH3 domain-supported polypeptide (CH3 domain "b") destabilize the association between CH3 domain "a" and a CH3 domain of another CH3 domain-supported polypeptide different from CH3 domain "b" (CH3 domain "c"). In other words, in some embodiments, modifications to CH3 domain "a" that stabilize the association between CH3 domain "a" and CH3 domain "b" destabilize the association between CH3 domain "a" and CH3 domain "c".

[0133] In some aspects and embodiments, interactions between specific CH3 domain-supported polypeptides are enhanced / facilitated / increased by introducing substitutions to the CH3 domains to stabilize the association between the CH3 domain of one CH3 domain-supported polypeptide and the CH3 domain of another CH3 domain-supported polypeptide.

[0134] In some embodiments, the destabilizing modification enhances / facilitates / increases the association between a CH3 domain containing the modification and another CH3 domain containing the destabilizing modification.

[0135] In some embodiments, the polypeptide complex according to the present disclosure comprises a polypeptide comprising a CH3 domain having complementary destabilizing modifications. <{

[0136] ]> In some embodiments, the amino acid residues introduced by the destabilizing modification of the CH3 domain of the polypeptide in the polypeptide complex according to the present disclosure form a protein-protein interaction in the association between the CH3 domains. The protein-protein interaction can be, for example, an electrostatic interaction (e.g., a salt bridge).

[0137] Illustratively, in relation to between a first CH3 domain containing the destabilizing modification E357K and a second CH3 domain containing the destabilizing modification K370E, a salt bridge is formed between the 357K NH3 + )>group and the 370E COO - group, and the interaction between the first CH3 domain and the second CH3 domain is stabilized. Similarly, in relation to between a first CH3 domain containing the destabilizing modification D356K and a second CH3 domain containing the destabilizing modification K439E, a salt bridge is formed between the 356K NH3 + group and the 439E COO - group, and the interaction between the first CH3 domain and the second CH3 domain is stabilized. <{

[0138] In some embodiments, the destabilizing modification of the first CH3 domain introduces an amino acid residue having a charge opposite to the charge of the amino acid residue introduced by the destabilizing modification of the second CH3 domain, wherein the amino acid residue introduced by the destabilizing modification of the first CH3 domain interacts in the association between the first CH3 domain and the second CH3 domain.

[0139] In some embodiments, the destabilization modification of the first CH3 domain introduces a positively charged amino acid residue, while the charge of the amino acid residue introduced by the destabilization modification of the second CH3 domain (which interacts with the first CH3 domain in association with the second CH3 domain) is negatively charged. In some embodiments, the destabilization modification of the first CH3 domain introduces a negatively charged amino acid residue, while the charge of the amino acid residue introduced by the destabilization modification of the second CH3 domain (which interacts with the first CH3 domain in association with the second CH3 domain) is positively charged.

[0140] For example, the modified E357K in the CH3 domain of human IgG1 destabilizes the association between a CH3 domain containing E357K and another CH3 domain containing E357K. Similarly, the modified K370E in the CH3 domain of human IgG1 destabilizes the association between a CH3 domain containing K370E and another CH3 domain containing K370E. However, modified E357K stabilizes the association with a CH3 domain containing modified K370E, and similarly, modified K370E stabilizes the association with 357K NH3 + Base and 370E COO - The formation of a salt bridge between the groups stabilizes the association with the CH3 domain, including the modified E357K.

[0141] Modifications to CH3 domains that promote association with other CH3 domains, and modifications that destabilize associations between CH3 domains, can be identified using assays for analyzing protein-protein interactions. In particular, such modifications can be identified using assays for analyzing the level of interaction between polypeptides containing CH3 domains.

[0142] Suitable techniques for identifying such modifications include resonance energy transfer techniques such as fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET), using appropriate label interaction partners, as described, for example, Ciruela, Curr. Opin. Biotechnol. (2008) 19(4):338-43. Other suitable techniques include protein-fragment complementation systems, such as NanoLuc and NanoBiT (described, for example, Thirukkumaran et al., Front. Chem. (2020) 7:938 and Dixon et al., ACS Chem. Biol. (2016) 11(2):400-408).

[0143] The candidate modification can be introduced into the amino acid sequence of the CH3 domain of a first polypeptide containing the CH3 domain, and the level of interaction between the first polypeptide and the second polypeptide containing the CH3 domain can be determined. The level of interaction between the first polypeptide (including the modification) and the second polypeptide can be compared to the level of interaction determined between the second polypeptide and a third polypeptide that is identical to the first polypeptide except for the lack of the candidate modification. A modification that results in a higher level of interaction between the first polypeptide and the second polypeptide than the level of interaction between the third polypeptide and the second polypeptide can be identified as a modification that promotes the association between the CH3 domain and the CH3 domain of the second polypeptide. A modification that results in a lower level of interaction between the first polypeptide and the second polypeptide than the level of interaction between the third polypeptide and the second polypeptide can be identified as a modification that destabilizes the association between the CH3 domain and the CH3 domain of the second polypeptide.

[0144] Modifications to CH3 domains to facilitate association with other CH3 domains, and modifications to destabilize associations between CH3 domains, can also be identified by analysis using EGAD software, as described, for example, in International Publication No. 2009 / 089004 A1 and Pokala, N. and Handel, TM, J. Mol. Biol. 347 (2005) 203-227, both of which are incorporated herein by reference in their entirety.

[0145] EGAD software can be used to estimate the binding free energy of a CH3-CH3 domain and to infer the effect of a given modification in a CH3 domain on the CH3-CH3 domain binding free energy. Briefly, the binding free energy of a given mutant CH3, including the modification, is defined as ΔΔGmut = μ(ΔGmut - ΔGwt)(mut = mutant, wt = wild type), where μ (generally 0.1) is a scaling factor used to normalize the predicted change in binding affinity so that it has a slope of 1 when compared to the experimental energy. The free energy of dissociation (ΔG) is defined as the energy difference between the complex (ΔGbound) and the free state (ΔGfree).

[0146] The destabilizing modifications described herein may be modifications to CH3 domains that increase the ΔG calculated according to EGAD (Pokala, N. and Handel, TM, J. Mol. Biol. 347 (2005) 203-227) compared to the ΔG calculated for equivalent unmodified CH3 domain interactions (when CH3 domain interaction pairs are introduced into CH3 domains). Conversely, modifications to promote association between CH3 domains may be modifications that decrease the ΔG calculated according to EGAD compared to the ΔG calculated for equivalent unmodified CH3 domain interactions (when CH3 domain interaction pairs are introduced into CH3 domains).

[0147] Modifications to CH3 domains to facilitate association with other CH3 domains, and modifications to destabilize associations between CH3 domains, can also be identified as described in International Publication 2020 / 216883 A1, which is incorporated herein by reference. See, in particular, the experimental examples in International Publication 2020 / 216883 A1.

[0148] It will be understood that the modification of the CH3 domain of the polypeptide according to this disclosure is typically selected according to the desired production of the payload-supported polypeptide complex by polypeptide exchange of the constituent polypeptides of the precursor polypeptide complex.

[0149] The CH3 domain of the polypeptides in a given precursor polypeptide complex may include modifications that promote association between polypeptides in the precursor polypeptide complex. For example, the polypeptides in a given precursor polypeptide complex may include complementary “knob” and “hole” modifications (described below). One or both polypeptides in the precursor polypeptide complex may further include destabilizing modifications to reduce the affinity of association between the first and second polypeptides, reduce the stability of the precursor polypeptide complex, or increase the tendency of the precursor polypeptide complex to dissociate. However, the destabilizing modifications do not completely prevent the interaction between polypeptides (and thus the formation of the precursor polypeptide complex). Rather, the destabilizing modifications inhibit / reduce / destabilize the interaction between polypeptides in a given precursor complex to such an extent that the affinity of the interaction between its constituent polypeptides is lower than the affinity of the interaction between the constituent polypeptides of the given precursor complex and the constituent polypeptides of a second different precursor complex. In this way, when the first and second polypeptide complexes are incubated with each other, polypeptide exchange occurs, yielding new polypeptide complexes containing one polypeptide each from the first and second precursor polypeptide complexes.

[0150] As an example, the first precursor polypeptide complex may include (i) a first polypeptide containing a CH3 domain with knob variant 366W and destabilization variant 370E; and (ii) a second polypeptide containing hole variants 407V, 366S, and 368A. The first and second polypeptides interact via their CH3 domains to form a polypeptide complex, but the glutamate residue at position 370 of the CH3 domain of the first polypeptide destabilizes the complex by introducing a charge repulsion between the glutamate residue at position 370 of the CH3 domain of the first polypeptide and the glutamate residue at position 357 of the CH3 domain of the second polypeptide (i.e., with respect to the stability of the complex that would be formed between the two polypeptides in the absence of the destabilization variant (i.e., if the residue at position 370 of the CH3 domain of the first polypeptide is a wild-type lysine residue of human IgG1)).

[0151] In such a situation, the second precursor polypeptide complex comprises (i) the first polypeptide containing a CH3 domain with knob variant 366W; and (ii) the second polypeptide containing hole variants 407V, 366S, 368A, and destabilization variant 357K. Here again, the first and second polypeptides interact via their CH3 domains to form a polypeptide complex, but the lysine residue at position 357 of the CH3 domain of the second polypeptide destabilizes the complex by introducing a charge repulsion between the lysine residue at position 357 of the CH3 domain of the second polypeptide and the lysine residue at position 370 of the CH3 domain of the first polypeptide (i.e., with respect to the stability of the complex that would be formed between the two polypeptides in the absence of the destabilization variant (i.e., when the residue at position 357 of the CH3 domain of the second polypeptide is a wild-type glutamate residue of human IgG1)).

[0152] In this scenario, polypeptide exchange between the two precursor polypeptide complexes is advantageous, particularly because the 370E residue of the CH3 domain of the first polypeptide in the first precursor polypeptide complex forms a salt crosslink with the 357K residue of the CH3 domain of the second polypeptide in the second precursor polypeptide complex.

[0153] Therefore, two new polypeptide exchange complexes are formed from the precursor complexes: (a) a polypeptide complex comprising the first polypeptide of the first precursor polypeptide complex and the second polypeptide of the second precursor polypeptide complex, and (b) a polypeptide complex comprising the second polypeptide of the first precursor polypeptide complex and the first polypeptide of the second precursor polypeptide complex.

[0154] Polypeptide complex (a) in the previous paragraph will be understood to be more stable / less prone to dissociation / formation by polypeptides that interact with higher affinity compared to either of the precursor polypeptide complexes. Similarly, polypeptide complex (b) in the previous paragraph will be more stable / less prone to dissociation / formation by polypeptides that interact with higher affinity compared to either of the precursor polypeptide complexes, because none of its constituent polypeptides contain modifications that destabilize their association.

[0155] Modifications to CH3 domains for destabilizing / stabilizing associations between CH3 domains carrying such modifications and certain other CH3 domains are described, for example, in International Publication 2019 / 077092 A1, Dengl et al., Nat.Commun. (2020) 11:4974 and International Publication 2020 / 216883 A1, all of which are incorporated herein by reference in their entirety.

[0156] In some embodiments, the destabilizing modification includes or consists of modifications at one or more of the following locations: 345, 347, 349, 351, 354, 356, 357, 360, 362, 364, 366, 368, 370, 390, 392, 394, 397, 399, 400, 401, 405, 407, 409, 439, or 441. Therefore, in some embodiments, the CH3 domain including the destabilizing modification includes modification at one or more of the following locations: 345, 347, 349, 351, 354, 356, 357, 360, 362, 364, 366, 368, 370, 390, 392, 394, 397, 399, 400, 401, 405, 407, 409, 439, or 441.

[0157] In some embodiments, the destabilizing modifications include or consist of: substitution of Q347 with a positively charged amino acid and K360 with a negatively charged amino acid; substitution of Y349 with a negatively charged amino acid; substitution of L351 with a hydrophobic amino acid and E357 with a hydrophobic amino acid; substitution of S364 with a hydrophobic amino acid; substitution of W366 with a hydrophobic amino acid and K409 with a negatively charged amino acid; substitution of L368 with a hydrophobic amino acid; substitution of K370 with a negatively charged amino acid; substitution of K370 with a negatively charged amino acid and K439 with a negatively charged amino acid; substitution of K392 with a negatively charged amino acid; substitution of T394 with a hydrophobic amino acid;

[0158] Substitution of V397 with a hydrophobic amino acid; substitution of D399 with a positively charged amino acid, and substitution of K409 with a negatively charged amino acid; substitution of S400 with a positively charged amino acid; substitution of F405W; Y407W; substitution of K439 with a negatively charged amino acid; substitution of S354 with a hydrophobic amino acid; substitution of D356 with a positively charged amino acid; substitution of E357 with a positively charged amino acid or a hydrophobic amino acid; substitution of D356 with a positively charged amino acid, and substitution of E357 with a positively charged amino acid or a hydrophobic amino acid Acid substitution; substitution of S364 with a hydrophobic amino acid; substitution of A368 with a hydrophobic amino acid; substitution of K392 with a negatively charged amino acid; substitution of T394 with a hydrophobic amino acid; substitution of D399 with a hydrophobic amino acid, and substitution of S400 with a positively charged amino acid; substitution of D399 with a hydrophobic amino acid, and substitution of F405 with a positively charged amino acid; substitution of V407 with a hydrophobic amino acid; substitution of K409 with a negatively charged amino acid; and substitution of K439 with a negatively charged amino acid.

[0159] In some embodiments, the CH3 domain including the knob modification includes, or comprises, the following destabilization modifications: substitution of Q347 with a positively charged amino acid and substitution of K360 with a negatively charged amino acid; substitution of Y349 with a negatively charged amino acid; substitution of L351 with a hydrophobic amino acid and substitution of E357 with a hydrophobic amino acid; substitution of S364 with a hydrophobic amino acid; substitution of W366 with a hydrophobic amino acid and substitution of K409 with a negatively charged amino acid; substitution of L368 with a hydrophobic amino acid; substitution of K370 with a negatively charged amino acid and substitution of K439 with a negatively charged amino acid; substitution of K392 with a negatively charged amino acid; substitution of T394 with a hydrophobic amino acid;

[0160] Substitution of V397 with a hydrophobic amino acid; substitution of D399 with a positively charged amino acid, and substitution of K409 with a negatively charged amino acid; substitution of S400 with a positively charged amino acid; substitution of F405W; Y407W; and substitution of K439 with a negatively charged amino acid.

[0161] In some embodiments, the CH3 domain including hole modifications includes, or comprises, destabilizing modifications: substitution of S354 with a hydrophobic amino acid; substitution of D356 with a positively charged amino acid; substitution of E357 with a positively charged or hydrophobic amino acid; substitution of D356 with a positively charged amino acid and substitution of E357 with a positively charged or hydrophobic amino acid; substitution of S364 with a hydrophobic amino acid; substitution of A368 with a hydrophobic amino acid; substitution of K392 with a negatively charged amino acid; substitution of T394 with a hydrophobic amino acid; substitution of D399 with a hydrophobic amino acid and substitution of S400 with a positively charged amino acid; substitution of D399 with a hydrophobic amino acid and substitution of F405 with a positively charged amino acid; substitution of V407 with a hydrophobic amino acid; substitution of K409 with a negatively charged amino acid; and substitution of K439 with a negatively charged amino acid.

[0162] In some embodiments, the CH3 domain including the destabilizing modification includes one or more of the following: 345R; 347K; 349W or 349E; 351F or 351Y; 354E or 354V; 356S, 356A or 356K; 357S, 357A, 357L, 357F or 357K; 360S or 360E; 362E; 364V or 364L; 366I; 368F or 368V; 370E; 390E; 392E or 392D; 394I; 397Y; 399A or 399K; 400K; 401R; 405W; 407W, 407L or 407I; 409D, 409E or 409I; 439E; or 441Y. In some embodiments, the CH3 domains including the destabilizing modification include 357S, 357A, 357L, 357F, or 357K (e.g., 357K). In some embodiments, the CH3 domains including the destabilizing modification include 370E. In some embodiments, the CH3 domains including the destabilizing modification include 356S, 356A, or 356K (e.g., 356K). In some embodiments, the CH3 domains including the destabilizing modification include 439E.

[0163] In some embodiments, the destabilizing modification is or includes: E345R; Q347K; Y349W or Y349E; L351F or L351Y; S354E or S354V; D356S, D356A or D356K; E357S, E357A, E357L, E357F or E357K; K360S or K360E; Q362 E; S364V or S364L; T366I; L368F or L368V; K370E; N390E; K392E or K392D; T394I; V397Y; D399A or D399K; S400K; D401R; F405W; Y407W, Y407L or Y407I; K409D, K409E or K409I; K439E; or L441Y. In some embodiments, the destabilizing modifier is or includes E357S, E357A, E357L, E357F or E357K (e.g., E357K). In some embodiments, the destabilizing modifier is or includes K370E. In some embodiments, the destabilizing modifier is or includes D356S, D356A, or D356K (e.g., D356K). In some embodiments, the destabilizing modifier is or includes K439E.

[0164] In some embodiments, the polypeptide complex according to the present disclosure comprises (i) a first polypeptide having a CH3 domain including 357S, 357A, 357L, 357F, or 357K (e.g., 357K), and (ii) a second polypeptide having a CH3 domain including 370E.

[0165] In some embodiments, the polypeptide complex according to the present disclosure comprises (i) a first polypeptide having a CH3 domain comprising 356S, 356A, or 356K (e.g., 356K), and (ii) a second polypeptide having a CH3 domain comprising 439E.

[0166] In some embodiments, the polypeptide complex according to the present disclosure comprises (i) a first polypeptide having a CH3 domain including destabilized modified E357S, E357A, E357L, E357F, or E357K (e.g., E357K), and (ii) a second polypeptide having a CH3 domain including destabilized modified K370E.

[0167] In some embodiments, the polypeptide complex according to the present disclosure comprises (i) a first polypeptide having a CH3 domain including destabilized variant D356S, D356A, or D356K (e.g., D356K), and (ii) a second polypeptide having a CH3 domain including destabilized variant K439E.

[0168] In some embodiments, the destabilizing modification attenuates / inhibits / reduces the association between CH3 domains, including modifications that promote their association. Such destabilizing modifications may be “reverse mutations” that return to amino acid residues of the CH3 domains in the absence of modifications that promote the association between CH3 domains.

[0169] In some embodiments, the destabilizing modification attenuates / inhibits / reduces the association between a CH3 domain containing a knob modification (e.g., a knob-cys modification) and a CH3 domain containing a hole modification (e.g., a hole-cys modification). Such a destabilizing modification may be a "reverse mutation" that returns to an amino acid residue of the CH3 domain in the absence of the knob / hole modification.

[0170] In some embodiments, the destabilizing modification disrupts / partially disrupts the knob structure in a CH3 domain containing a knob modification. In some embodiments, the destabilizing modification disrupts / partially disrupts the hole structure in a CH3 domain containing a hole modification. In some embodiments, the destabilizing modification to a CH3 domain containing a knob or hole modification can partially or completely remove the knob / hole modification. In some embodiments, a CH3 domain containing a knob or hole modification and further containing a destabilizing modification may have the amino acid sequence of a CH3 domain lacking the knob / hole modification.

[0171] In some embodiments, the CH3 domain, which includes a knob modification and further includes a destabilization modification, does not include W at position 366 and / or does not include Y. In some embodiments, the CH3 domain, which includes a knob modification and further includes a destabilization modification, includes 366T.

[0172] In some embodiments, a CH3 domain including a hole modification and further including a destabilizing modification does not contain V at position 407, and / or A, and / or S, and / or T. In some embodiments, a CH3 domain including a hole modification and further including a destabilizing modification contains 407Y.

[0173] In some embodiments, a CH3 domain including a hole modification and further including a destabilization modification does not contain S at position 366 and / or V and / or A. In some embodiments, a CH3 domain including a hole modification and further including a destabilization modification contains 366T.

[0174] In some embodiments, a CH3 domain including a hole modification and further including a destabilizing modification does not contain A at position 368, and / or V, and / or S, and / or T. In some embodiments, a CH3 domain including a hole modification and further including a destabilizing modification includes 368L.

[0175] In some embodiments, the CH3 domain, which includes a knob-cys modification and further includes a destabilizing modification, does not contain C at position 354. In some embodiments, the CH3 domain, which includes a knob-cys modification and further includes a destabilizing modification, contains 354S.

[0176] In some embodiments, the CH3 domain, which includes a hole-cys modification and further includes a destabilizing modification, does not contain C at position 349. In some embodiments, the CH3 domain, which includes a hole-cys modification and further includes a destabilizing modification, contains 349Y.

[0177] In some embodiments, the polypeptide complex according to this disclosure is a polypeptide having a CH3 domain comprising (i) a knob modification and at least one modification selected from the following: Y349E; Y349D; S364V; S364I; S364L; L368F; K370E; K370D; K392E; K392D; T394L; T394I; V397Y; S400K; S400R; F405W; Y407W; K349E; K439D; Q347K and K360E; Q347R; K360E; Q347K and K360D; Q347R and K360D; L351F and E357F; W366I and K409E; W366L and K409E; W366K and K4 polypeptides having a CH3 domain including (ii) hole modification and at least one modification selected from the following: S354V; S354I; S354I; S35 Includes 4L;D356K;D356R;E357K;E357R;E357F;S364L;S364I;A368F;K392D;K392E;T394L;T394I;V407Y;K409E;K409D;K439D;K439E;D399A and S400K;D399A and S400R;D399A and F405W.

[0178] In some embodiments, the polypeptide complex according to this disclosure is a polypeptide having a CH3 domain comprising (i) a knob modification and at least one modification selected from the following: Y349E; S364V; L368F; K370E; K392D; T394I; V397Y; S400K; F405W; Y407W; K349E; Q347K and K360E; L351F and E357F; W Polypeptides having a CH3 domain, including (ii) hole modification and at least one modification selected from the following: S354V; D356K; E357K; E357F; S364L; A368F; K392E; T394I; V407Y; K409E; K439E; and D399A and S400K.

[0179] In some embodiments, the polypeptide complex according to this disclosure is a polypeptide having a CH3 domain comprising (i) a knob modification and at least one modification selected from the following: Y349E; S364V; L368F; K370E; K392D; T394I; V397Y; S400K; F405W; Y407W; K349E; Q347K and K360E; L351F and E357F; W366I and K409E; and D399K and K409E; and (ii) hole modification, and polypeptides having a CH3 domain including at least one modification selected from the following: D356K; D356R; E357K; E357R; E357F; S364L; S364I; V407Y; K409E; K409D; D399A and S400K; and D399A and S400R.

[0180] In some embodiments, the polypeptide complexes according to the present disclosure include (i) polypeptides having a CH3 domain comprising a knob modification and at least one modification selected from the following: Y349E; K370E; K392D; T394I; V397Y; F405W; Y407W; K349E; Q347K and K360E; W366I and K409E; and D399K and K409E; and (ii) polypeptides having a CH3 domain comprising a hole modification and at least one modification selected from the following: D356K; E357K; E357F; S364L; V407Y; K409E; and D399A and S400K.

[0181] In some embodiments, the polypeptide complex according to the present disclosure comprises (i) a polypeptide having a CH3 domain comprising a knob modification (e.g., T366W or T366W and S354C) and one modification from row 1 to 49 of column A in Table II; and (ii) a polypeptide having a CH3 domain comprising a hole modification (e.g., Y407V, T366S and L368A; or Y407V, T366S, L368A and Y349C) and one modification from row 1 to 49 of column B in Table II (e.g., Y407V, T366S and L368A; or Y407V, T366S, L368A and Y349C). [table] TIFF2026509081000004.tif253170TIFF2026509081000005.tif109170

[0182] In preferred embodiments, for a given polypeptide complex, CH3 domain modifications including knob modifications from column A and CH3 domain modifications including hole modifications from column B are selected from the same row in Table II. For example, in some embodiments, the polypeptide complex according to the present disclosure comprises (i) a polypeptide having a CH3 domain including a knob modification (e.g., T366W or T366W and S354C) and modifications K370E and K439E (row 1 of column A); and (ii) a polypeptide having a CH3 domain including a hole modification (e.g., Y407V, T366S and L368A; or Y407V, T366S, L368A and Y349C) and modification D356K (row 1 of column B).

[0183] In some embodiments, the polypeptide complex according to the present disclosure comprises (i) a polypeptide having a CH3 domain including a knob modification (e.g., T366W or T366W and S354C) and one modification from rows 1 to 12 of column A in Table III; and (ii) a polypeptide having a CH3 domain including a hole modification (e.g., Y407V, T366S and L368A; or Y407V, T366S, L368A and Y349C) and one modification from rows 1 to 12 of column B in Table III (in preferred embodiments, for a given polypeptide complex, the CH3 domain modification including the knob modification from column A and the CH3 domain modification including the hole modification from column B are selected from the same row in Table III). [table] TIFF2026509081000006.tif99170

[0184] In some embodiments, a CH3 domain including a knob modification either does not include an destabilizing modification or includes at least one modification selected from: K370E; K370D; K392E; K392D; V397Y; K370E and K439E; K370D and K439E; K370E and K439D; and K370D and K439D; and a CH3 domain including a hole modification includes at least one modification selected from: E357K; E357R; S364L; S364I; V407Y; V407F and A368F.

[0185] In some embodiments, a CH3 domain including a knob modification includes at least one modification selected from: K370E; K392D; V397Y; K370E and K439E; and a CH3 domain including a hole modification includes at least one modification selected from: E357K; S364L; V407Y; and A368F.

[0186] In some embodiments, a CH3 domain including a knob modification includes at least one modification selected from: K370E; K370D; K392E; K392D; V397Y; K370E and K439E; K370D and K439E; K370E and K439D; and K370D and K439D; and a CH3 domain including a hole modification includes at least one modification selected from: E357K; E357R; S364L; S364I; V407Y; and V407F.

[0187] In some embodiments, a CH3 domain including a knob modification includes at least one modification selected from: K370E; K392D; V397Y; and K370E and K439E; and a CH3 domain including a hole modification includes at least one modification selected from: E357K; S364L; and V407Y.

[0188] In some embodiments, the polypeptide complex according to the present disclosure comprises (i) a polypeptide having a CH3 domain including a knob modification (e.g., T366W or T366W and S354C) and one modification from rows 1 to 14 of column A in Table IV; and (ii) a polypeptide having a CH3 domain including a hole modification (e.g., Y407V, T366S and L368A; or Y407V, T366S, L368A and Y349C) and one modification from rows 1 to 14 of column B in Table IV (in preferred embodiments, for a given polypeptide complex, the CH3 domain modification including the knob modification from column A and the CH3 domain modification including the hole modification from column B are selected from the same row in Table IV). [table] TIFF2026509081000007.tif112170

[0189] In some embodiments, the polypeptide complex according to this disclosure comprises (i) a polypeptide having a CH3 domain including a knob modification (e.g., T366W or T366W and S354C) and one modification from rows 1 to 8 of column A in Table V; and (ii) a polypeptide having a CH3 domain including a hole modification (e.g., Y407V, T366S and L368A; or Y407V, T366S, L368A and Y349C) and one modification from rows 1 to 8 of column B in Table V (in preferred embodiments, for a given polypeptide complex, the CH3 domain modification including the knob modification from column A and the CH3 domain modification including the hole modification from column B are selected from the same row in Table V): [table] TIFF2026509081000008.tif72170

[0190] In some embodiments, the polypeptide complex according to the Disclosure (in particular, the precursor polypeptide complex according to the Disclosure) comprises (i) a polypeptide having a CH3 domain including a knob modification (e.g., T366W or T366W and S354C) and one modification from rows 1 to 18 of column A in Table VI; and (ii) a polypeptide having a CH3 domain including a hole modification (e.g., Y407V, T366S and L368A; or Y407V, T366S, L368A and Y349C) and one modification from rows 1 to 18 of column B in Table VI (in preferred embodiments, for a given polypeptide complex, the CH3 domain modification including the knob modification from column A and the CH3 domain modification including the hole modification from column B are selected from the same row in Table VI). [table] TIFF2026509081000009.tif158170

[0191] Precursor polypeptide complexes containing polypeptides with CH3 domains that include such combinations of destabilizing modifications exhibit particularly beneficial polypeptide chain exchange.

[0192] In some embodiments, the polypeptide complex according to the Disclosure (in particular, the precursor polypeptide complex according to the Disclosure) comprises (i) a polypeptide having a CH3 domain including a knob modification (e.g., T366W or T366W and S354C) and one modification from rows 1 to 24 of column A in Table VII; and (ii) a polypeptide having a CH3 domain including a hole modification (e.g., Y407V, T366S and L368A; or Y407V, T366S, L368A and Y349C) and one modification from rows 1 to 24 of column B in Table VII (in preferred embodiments, for a given polypeptide complex, the CH3 domain modification including the knob modification from column A and the CH3 domain modification including the hole modification from column B are selected from the same row in Table VII). [table] TIFF2026509081000010.tif203170

[0193] Precursor polypeptide complexes containing polypeptides with CH3 domains that include such combinations of destabilizing modifications exhibit particularly beneficial polypeptide chain exchange.

[0194] Further possible modifications to the CH3 domain In some aspects and embodiments, the CH3 domain of the Disclosure includes modifications to alter the Fc-mediated function.

[0195] Modifications to the antibody Fc region that affect Fc-mediated function are known in the art, such as those described in Wang et al., Protein Cell (2018) 9(1):63-73, and are incorporated herein by reference in their entirety. Exemplary Fc region modifications known to affect antibody effector function are summarized in Table 1 of Wang et al., Protein Cell (2018) 9(1):63-73.

[0196] In some embodiments, the CH3 domain of the present disclosure, when provided to an Fc region containing the CH3 domain, includes modifications that increase or decrease the level of Fc-mediated function (i.e., compared to the level of Fc-mediated function shown by an equivalent Fc region without modifications).

[0197] In some embodiments, the CH3 domain includes modifications to increase Fc-mediated function. In some embodiments, the CH3 domain includes modifications to increase binding to ADCC, ADCP, and / or CDC. In some embodiments, the CH3 domain includes modifications to increase binding to Fc receptors (e.g., Fcγ receptors, one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb). In some embodiments, the CH3 domain includes modifications to increase binding to FcRn. In some embodiments, the CH3 domain includes modifications to increase binding to complement proteins (e.g., C1q).

[0198] In some embodiments, the CH3 domain includes modifications to reduce Fc-mediated function. In some embodiments, the CH3 domain includes modifications to reduce ADCC, ADCP, and / or CDC. In some embodiments, the CH3 domain includes modifications to reduce binding to Fc receptors (e.g., Fcγ receptors, one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb). In some embodiments, the CH3 domain includes modifications to reduce binding to FcRn. In some embodiments, the CH3 domain includes modifications to reduce binding to complement proteins (e.g., C1q).

[0199] In embodiments and aspects of the present disclosure, the CH3 domain (for example, the CH3 domain of the constituent polypeptide of the polypeptide complex according to the present disclosure) includes CH3 domain modifications known to affect Fc-mediated function as described in Wang et al., Protein Cell (2018) 9(1):63-73.

[0200] Payload section The aspects and embodiments of this disclosure relate to polypeptide complexes including a payload portion, and polypeptides including a payload portion.

[0201] In this specification, “payload portion” refers to any portion provided to a polypeptide complex. In some embodiments, the payload portion is or includes a detectable portion (e.g., a fluorescent portion, a luminescent portion, a radiopaque / contrast agent, a radiolabeled portion, an immunodetectable portion), a portion having detectable activity (e.g., an enzyme portion), or a drug portion (e.g., a cytotoxic portion).

[0202] In some embodiments, the payload portion is or includes a detectable portion. “Detectable portion” means a portion capable of producing a detectable signal indicating its presence. A detectable portion includes portions detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means.

[0203] In some embodiments, the payload portion is or includes a fluorescent portion. Fluorescent portions are well known in the art and include, for example, fluorescein, rhodamine, tetramethylrhodamine, allophycocyanin, phycoerytherin, phycocyanin, cyanines (e.g., Cy2, Cy3, Cy3B, Cy3.5, C5, Cy5.5, Cy7), 4-methylumbelliferone, 7-amino-4-methylcoumarin, o-phthalaldehyde, fluorescein, eosin, nitrobenzoxadiazole, Texas Red, green fluorescent protein (GFP), and chelates of rare earth metals such as europium (Eu), terbium (Tb), and samarium (Sm).

[0204] In some embodiments, the payload portion is or includes a luminescent portion. Examples of luminescent portions include radiant luminescent portions (e.g., radium, promethium, tritium), chemiluminescent portions (e.g., acridinium esters, imidazoles, acridinium salts, oxalate esters, acridinium esters, luminol, isoluminol), and bioluminescent portions (e.g., luciferin (e.g., coelenterazine), aequorin).

[0205] In some embodiments, the payload portion is or contains radiopaque or contrast agents. Examples of such agents include barium, diatrizoate, ethiozide oil, gallium citrate, iocarminic acid, iosetamic acid, iodoamide, iodipamide, iodoxamic acid, ioglamid, iohexol, iopamidol, iofanic acid, ioprosemic acid, iosefamic acid, ioseric acid, iosramide meglumine, iosemetic acid, iotasul, iotheric acid, iotalamic acid, iotroxic acid, ioxagric acid, ioxotrizoic acid, ipodate, meglumine, metrizamide, metrizoate, propyliodone, and thallium chloride.

[0206] In some embodiments, the payload portion is or includes a radioactive label. Hydrogen is used as the radioactive label. 3 ,sulfur 35 , carbon-14 14 , Lin 32 iodine 123 iodine 125 iodine 126 iodine 131 iodine 133 ,bromine 77 ,technetium 99m ,indium 111 ,indium 113m ,gallium 67 ,gallium 68 ,ruthenium 95 ,ruthenium 97 ,ruthenium 103 ,ruthenium 105 ,mercury 207 ,mercury 203 ,rhenium 99m ,rhenium 101 ,rhenium 105 ,scandium 47 , Tellurium 121m , Tellurium 122m , Tellurium 125m ,thulium 165 ,thulium 167 ,thulium 168 ,copper 67 fluorine 18 ,yttrium 90 ,palladium 100 bismuth 217 and antimony 211 Examples of radioactive isotopes include the following.

[0207] In some embodiments, the payload portion is or includes an immunodetectable portion. The immunodetectable portion is a portion that can be detected using immunological techniques, for example, using an antibody. Immunodetectable portions include, for example, peptide / polypeptide epitope tags, antibodies, receptors, ligands, and nucleic acids. Examples of immunodetectable portions include epitope tags such as 6xHis, FLAG, c-Myc, StrepTag, hemagglutinins, calmodulin-binding proteins (CBPs), glutathione-s-transferase (GST), maltose-binding proteins (MBPs), thioredoxins, S-peptides, T7 peptides, SH2 domains, avidins, streptavidins, and haptens (e.g., biotin, digoxigenin, dinitrophenol).

[0208] In some embodiments, the payload portion is or includes a portion having detectable activity, such as an enzyme portion. Enzyme portions include, for example, luciferase, glucose oxidase, galactosidase (e.g., beta-galactosidase), glucolinidase, phosphatase (e.g., alkaline phosphatase), peroxidase (e.g., horseradish peroxidase), and cholinesterase.

[0209] In some embodiments, the payload portion is or includes a drug portion. The drug portion may be a chemical portion for providing a therapeutic effect. The drug portion may be a small molecule (e.g., an organic compound with a low molecular weight (<1000 daltons, typically about 300-700 daltons)). The drug portion is described, for example, in Parslow et al., Biomedicines. (2016) Sep;4(3):14 (the whole of which is incorporated herein by reference). In some embodiments, the drug portion may be or include a cytotoxic agent. In some embodiments, the drug portion may be or include a chemotherapeutic agent. Examples of drug portions include calicheamicin, DM1, DM4, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), SN-38, doxorubicin, duocalmycin, D6.5, and PBD.

[0210] In some embodiments, the payload portion does not contain or is not derived from the VH domain and / or VL domain of the antibody. In some embodiments, the payload portion does not contain or is not derived from the antibody or antibody fragment / derivative. In some embodiments, the payload portion does not contain or is not derived from the target binding moiety.

[0211] The payload portion of the Disclosure may be attached to / conjugated to the polypeptide of the Disclosure (e.g., the polypeptide of the polypeptide complex of the Disclosure) by any suitable means known in the Art.

[0212] In some embodiments, the payload portion may be covalently bonded to the polypeptide of the Disclosure (e.g., chemically conjugated). Chemical conjugation may be carried out by any suitable means, and those skilled in the art will be well aware of suitable techniques, but are not limited to: (1) direct coupling via protein functional groups (e.g., thiol-thiol bonds, amine-carboxyl bonds, amine-aldehyde bonds; enzymatic direct coupling); (2) homobifunctional coupling of amines (e.g., using bisaldehyde); (3) homobifunctional coupling of thiols (e.g., using bismaleimide); (4) homobifunctional coupling via photoactivators; (5) heterobifunctional coupling of amines to thiols (6) bifunctional coupling (e.g., using maleimide); (7) heterobifunctional coupling via photoactivating reagents (e.g., β-carbonyldiazo family); (8) introduction of amine-reactive groups into polysaccharides or oligosaccharides via cyanide bromide activation or carboxymethylation; (9) introduction of thiol-reactive groups into polysaccharides or oligosaccharides via heterobifunctional compounds such as maleimide-hydrazide; (10) protein-lipid conjugation via introduction of hydrophobic groups into proteins; and (11) protein-lipid conjugation via incorporation of reactive groups into lipids. Heterobifunctional "non-covalent coupling" techniques such as biotin-avidin interactions are also being considered. Conjugation techniques have been reviewed, for example, in Kalia and Raines, Curr. Org. Chem. (2010) 14(2):138-147, which is incorporated herein by reference in its entirety.

[0213] In some embodiments, the payload portion is attached to the polypeptide of this disclosure via a linker portion. Linker portions for linking the payload portion to the polypeptide (e.g., antibody polypeptides) are known in the art and have been reviewed, for example, in Su et al., Acta Pharm. Sin. B. (2021) 11(12):3889-3907, which is incorporated herein by reference in its entirety. The linker portion may be a cleavable linker portion (e.g., an acid-cleavable linker portion) or an incleavable linker portion.

[0214] In some embodiments, the payload portion is attached to the polypeptide of the Disclosure in a non-specific manner.

[0215] In some embodiments, the payload portion is covalently bonded to the side chain of an amino acid residue of the polypeptide. In some embodiments, the payload portion is covalently bonded to an amino acid having a side chain containing an amino group, such as lysine. In some embodiments, attachment may be via a peptide bond formed by a reaction between the amino group of the amino acid having a side chain containing an amino group and the N-hydroxysuccinimide (NHS) portion of the payload portion or linker portion. Exemplarily, in Example 2.2 of the present disclosure, the dye portion is conjugated to the polypeptide of the present disclosure via an NHS-lysine conjugation. Further exemplarily, in Example 2.3 of the present disclosure, the horseradish peroxidase portion is conjugated to the polypeptide of the present disclosure via an NHS-lysine conjugation.

[0216] In some embodiments, the payload portion is attached to the polypeptide of the Disclosure in a site-specific manner. In some embodiments, the payload portion is attached to (a) specific amino acids of the polypeptide. In some embodiments, the payload portion is attached to the N-terminus and / or C-terminus of the polypeptide.

[0217] In some embodiments, the payload portion is provided as a fusion polypeptide with the polypeptide of the Disclosure. That is, the amino acid sequence encoding the payload portion may be fused to the N-terminus and / or C-terminus of the polypeptide via peptide bonds. For example, in Example 2.5 of the Disclosure, the GFP portion is provided as a fusion polypeptide to the C-terminus of the CH3 domain of the polypeptide of the Disclosure.

[0218] In some embodiments, the payload portion is attached via crosslinking of lysine ε-amino and glutamine γ-carboxyamide groups. For example, in Example 2.4 of this disclosure, a ruthenium portion linked to a K tag is attached to a polypeptide containing a Q tag at its C-terminus via a transglutaminase-catalyzed aminotransfer reaction.

[0219] In some embodiments, the payload portion is attached, for example, in a site-specific manner, via the action of an enzyme for introducing the payload portion. For example, in Example 2.4 of this disclosure, a biotin ligase is used to introduce the biotin moiety to a lysine residue of the Avi tag provided at the N-terminus of the polypeptide.

[0220] In some embodiments, the polypeptide of the Disclosure does not include a payload portion. In some embodiments, the polypeptide complex of the Disclosure does not include a payload portion.

[0221] In some embodiments, the polypeptide complex of the present disclosure (e.g., the final payload-carrying polypeptide complex) comprises (i) a polypeptide containing a payload portion and (ii) a polypeptide not containing a payload portion. For example, in the final payload-carrying polypeptide complex (i.e., the “defined labeled antibody”) shown in the schematic diagram of Figure 1, one polypeptide of the complex (i.e., the polypeptide derived from the “donor” precursor complex) contains a payload portion, and the other polypeptide of the complex (i.e., the polypeptide derived from the “acceptor” precursor complex) does not contain a payload portion.

[0222] In some embodiments, the polypeptide or polypeptide complex according to the Disclosure comprises two or more payload portions. In some embodiments, the polypeptide or polypeptide complex according to the Disclosure comprises one or more payload portions (e.g., one of one, two, three, four or more). According to such embodiments, each payload portion may independently be a payload portion as defined above herein.

[0223] Functional part The aspects and embodiments of this disclosure relate to polypeptide complexes containing functional moieties and polypeptides containing functional moieties.

[0224] In this specification, “functional portion” refers to any portion having a function. In some embodiments, the functional portion is or includes a binding portion (e.g., an antibody or its target-binding fragment or derivative, a target-binding peptide / polypeptide, a target-binding nucleic acid), a detectable portion (e.g., a fluorescent portion, a luminescent portion, a radiopaque / contrast agent, a radiolabel, an immunodetectable portion), a portion having detectable activity (e.g., an enzyme portion), or a drug portion (e.g., a cytotoxic portion).

[0225] It will be understood that there is considerable overlap between the functional portion and the payload portion. In preferred embodiments, in polypeptides and / or polypeptide complexes according to this disclosure, which include a functional portion and a payload portion, the functional portion and the payload portion are not identical.

[0226] In some embodiments, the functional portion is or includes a binding portion. The binding portion includes, for example, an antibody (e.g., a monoclonal antibody, e.g., a multispecific antibody) that exhibits binding to a target molecule of interest, an antibody fragment / derivative (e.g., Fv, scFv, Fab, scFab, F(ab')2, Fab2, diabody, triabody, scFv-Fc, minibody, single-domain antibody (e.g., VhH), etc.) that exhibits binding to a target molecule of interest. The target binding portion includes a portion containing an antigen-binding domain derived from the antibody, e.g., the Fv (e.g., provided as scFv) or Fab region of the antibody, or the entire antibody, or a portion consisting of these.

[0227] In some embodiments, the target-binding moiety includes an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) having the ability to specifically bind to a target antigen. The antigen-binding domain formed by VH and VL may also be referred to herein as the Fv region. In some embodiments, the target-binding moiety includes or consists of an antigen-binding polypeptide or an antigen-binding polypeptide complex. An antigen-binding polypeptide complex may include two or more polypeptides that together form an antigen-binding domain. The polypeptides of the antigen-binding polypeptide complex may associate covalently or non-covalently. In some embodiments, polypeptides form part of a larger polypeptide containing polypeptides (e.g., in the case of scFv containing VH and VL, or in the case of scFab containing VH-CH1 and VL-CL). In some embodiments, polypeptides are not provided in the same polypeptide (e.g., in the case of Fab fragments or IgG-like portions).

[0228] In some embodiments, the target-binding moiety includes or consists of an aptamer capable of binding to the target antigen, such as a nucleic acid aptamer (reviewed, for example, in Zhou and Rossi, Nat. Rev. Drug. Discov. (2017) 16(3):181-202). In some embodiments, the target-binding moiety includes or consists of target-binding peptides / polypeptides, e.g., peptide aptamers, thioredoxin, monobodies, anticharin, Kunitz domains, avimers, noctin, finomers, atrimers, DARPin, afibodies, nanobodies (i.e., single-domain antibodies (sdAbs)), affilins, armadillo repeat proteins (ArmRPs), OBody, or fibronectin—for example, reviewed in Reverdatto et al., Curr. Top. Med. Chem. (2015) 15(12):1082-1101, the whole of which is incorporated herein by reference (see also, e.g., Boersma et al., J. Biol. Chem. (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48).

[0229] In some embodiments, the functional portion is or includes a fluorescent portion, for example, as described above in this specification. In some embodiments, the functional portion is or includes a luminescent portion, for example, as described above in this specification. In some embodiments, the functional portion is or includes a radiopaque or contrast agent, for example, as described above in this specification. In some embodiments, the functional portion is or includes a radiolabel, for example, as described above in this specification. In some embodiments, the functional portion is or includes an immunodetectable portion, for example, as described above in this specification. In some embodiments, the functional portion is or includes a portion having detectable activity, for example, an enzyme portion, for example, as described above in this specification. In some embodiments, the functional portion is or includes a drug portion, for example, as described above in this specification.

[0230] The functional portion may be attached to / conjugated to the polypeptide of the Disclosure (e.g., the polypeptide of the polypeptide complex of the Disclosure) by any suitable means, including the means described above in this specification with respect to the payload portion. In some embodiments, the functional portion may be covalently bonded to the polypeptide of the Disclosure (e.g., chemically conjugated). In some embodiments, the functional portion is attached to the polypeptide via a linker portion.

[0231] In some embodiments, the functional moiety is attached to the polypeptide of the Disclosure in a site-specific manner. In some embodiments, the functional moiety is attached to (a) a specific amino acid of the polypeptide. In some embodiments, the functional moiety is attached to the N-terminus and / or C-terminus of the polypeptide.

[0232] In some embodiments, the functional moiety is provided as a fusion polypeptide with the polypeptide of the Disclosure. That is, the amino acid sequence encoding the functional moiety may be fused to the N-terminus and / or C-terminus of the polypeptide via peptide bonds. For example, in the experimental example molecule, the functional moiety comprises a Fab fragment fused to the N-terminus of the CH3 domain-supported polypeptide of the Disclosure, and its VH-CH1 component is provided as a fusion protein. It will be understood that the VL-CL component of the Fab fragment associates through an interaction between the VH-CH1 region and the VL-CL region.

[0233] In some embodiments, the functional moiety is attached to the polypeptide in a non-specific manner (for example, via covalent bonding to the side chains of amino acid residues of the polypeptide (e.g., amino acids having side chains containing an amino group, e.g., lysine)). In some embodiments, the functional moiety is attached to the amino groups of amino acid residues of the polypeptide via peptide bonds formed by the reaction of the NHS moiety of the functional moiety or linker moiety.

[0234] In some embodiments, the polypeptides of the Disclosure do not include a functional moiety. In some embodiments, the polypeptide complexes of the Disclosure do not include a functional moiety.

[0235] In some embodiments, the polypeptide complex of the present disclosure (e.g., the final functionally supported polypeptide complex) comprises (i) a polypeptide containing a functional moiety and (ii) a polypeptide not containing a functional moiety. For example, in the final functionally supported polypeptide complex (i.e., the “defined labeled antibody”) shown in the schematic diagram of Figure 1, one polypeptide of the complex (i.e., the polypeptide derived from the “acceptor” precursor complex) contains a functional moiety (i.e., the Fab region), and the other polypeptide of the complex (i.e., the polypeptide derived from the “donor” precursor complex) does not contain a functional moiety.

[0236] In some embodiments, the polypeptide or polypeptide complex according to the Disclosure comprises two or more functional parts. In some embodiments, the polypeptide or polypeptide complex according to the Disclosure comprises one or more functional parts (e.g., one of one, two, three, four or more). According to such embodiments, each functional part may independently be a functional part as defined above herein.

[0237] Further domains / areas of polypeptides In aspects and embodiments of this disclosure, the polypeptides described herein (e.g., constituent polypeptides of the polypeptide complex of this disclosure) include further amino acid / sequences of amino acids, e.g., amino acid / amino acid sequences that form further peptide / polypeptide / domains.

[0238] CH2 domain The polypeptides according to this disclosure may include a CH2 domain. The CH2 domain may be provided upstream of the CH3 domain (i.e., at the N-terminus) in the amino acid sequence of the polypeptide. In such embodiments, the CH2 domain may be provided immediately upstream of the CH3 domain (i.e., adjacent to the CH3 domain in the amino acid sequence of the polypeptide).

[0239] In this specification, the "CH2 domain" refers to the amino acid sequence corresponding to the CH2 domain of immunoglobulin (Ig). The CH2 domain is the region of Ig formed by positions 231-340 of the immunoglobulin constant domain according to the EU numbering system described in Edelman et al., Proc. Natl. Acad. Sci. USA (1969) 63(1):78-85.

[0240] In some embodiments, the CH2 domain corresponds to or is derived from the CH2 domain of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM.

[0241] In some embodiments, the CH2 domain corresponds to or is derived from the CH2 domain of human IgG (e.g., hIgG1, hIgG2, hIgG3, hIgG4), hIgA (e.g., hIgA1, hIgA2), hIgD, hIgE, or hIgM. In some embodiments, the CH2 domain corresponds to or is derived from the CH2 domain of human IgG1 allotype (e.g., G1m1, G1m2, G1m3, or G1m17).

[0242] Sequence ID 11 shows the CH2 domains of the G1m1 and G1m3 allotypes of human IgG1. Sequence ID 12 shows the CH2 domain of human IgG2. Sequence ID 13 shows the CH2 domain of human IgG3. Sequence ID 14 shows the CH2 domain of human IgG4. Sequence ID 15 shows the second Ig-like domain of human IgA1. Sequence ID 16 shows the second Ig-like domain of human IgA2. Sequence ID 17 shows the second Ig-like domain of human IgD. Sequence ID 18 shows the second Ig-like domain of human IgE. Sequence ID 19 shows the CH2 domain of human IgM.

[0243] In some embodiments, the CH2 domain according to the present disclosure comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, or 19, for example, one of the following amino acid sequence identity values: ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%.

[0244] In some embodiments, the CH2 domain includes or comprises an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NOs: 11, 12, 13, or 14, for example, one of the following amino acid sequence identity levels: ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%.

[0245] In some embodiments, the CH2 domain includes or comprises an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO: 11, for example, one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity.

[0246] In some embodiments, the CH2 domains of the Disclosure include modifications to facilitate association with other CH2 domains. In some embodiments, the CH2 domains include modifications to facilitate heteromerization, i.e., association between non-identical CH2 domains.

[0247] If the CH2 region described herein includes modifications and further includes or consists of amino acid sequences within a certain threshold percentage amino acid sequence identity with respect to the reference amino acid sequence, it will be understood that any mutations with respect to the reference amino acid sequence are limited to reference sequence locations other than the modified location.

[0248] In some embodiments, the CH2 domain of the present disclosure, when provided to an Fc region containing the CH2 domain, includes modifications that increase or decrease the level of Fc-mediated function (i.e., compared to the level of Fc-mediated function shown by an equivalent Fc region without modifications).

[0249] In some embodiments, the CH2 domain includes modifications to increase Fc-mediated function. In some embodiments, the CH2 domain includes modifications to increase binding to ADCC, ADCP, and / or CDC. In some embodiments, the CH2 domain includes modifications to increase binding to Fc receptors (e.g., Fcγ receptors, one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb). In some embodiments, the CH2 domain includes modifications to increase binding to FcRn. In some embodiments, the CH2 domain includes modifications to increase binding to complement proteins (e.g., C1q).

[0250] In some embodiments, the CH2 domain includes modifications to reduce Fc-mediated function. In some embodiments, the CH2 domain includes modifications to reduce ADCC, ADCP, and / or CDC. In some embodiments, the CH2 domain includes modifications to reduce binding to Fc receptors (e.g., Fcγ receptors, one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb). In some embodiments, the CH2 domain includes modifications to reduce binding to FcRn. In some embodiments, the CH2 domain includes modifications to reduce binding to complement proteins (e.g., C1q).

[0251] In embodiments and aspects of the present disclosure, the CH2 domain (for example, the CH2 domain of the constituent polypeptide of the polypeptide complex according to the present disclosure) includes CH2 domain modifications known to affect Fc-mediated function, as described in Wang et al., Protein Cell (2018) 9(1):63-73.

[0252] Hinge area Polypeptides according to this disclosure may include a hinge region. The hinge region may be provided upstream (i.e., at the N-terminus) of the CH2 domain (and CH3 domain) in the amino acid sequence of the polypeptide. In such embodiments, the hinge region may be provided immediately upstream of the CH2 domain (i.e., adjacent to the CH2 domain in the amino acid sequence of the polypeptide).

[0253] In this specification, “hinge region” refers to the amino acid sequence corresponding to the hinge region of immunoglobulin (Ig). The hinge region is the region of Ig formed by positions 216-230 of the immunoglobulin constant domain according to the EU numbering system described in Edelman et al., Proc. Natl. Acad. Sci. USA (1969) 63(1):78-85.

[0254] In some embodiments, the hinge region corresponds to or is derived from the hinge region of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE or IgM.

[0255] In some embodiments, the hinge region corresponds to or is derived from the hinge region of human IgG (e.g., hIgG1, hIgG2, hIgG3, hIgG4), hIgA (e.g., hIgA1, hIgA2), hIgD, hIgE or hIgM. In some embodiments, the hinge region corresponds to or is derived from the hinge region of a human IgG1 allotype (e.g., G1m1, G1m2, G1m3 or G1m17).

[0256] The hinge regions of the G1m1 and G1m3 allotypes of human IgG1 are shown in SEQ ID NO: 20. The hinge region of human IgG{2} is shown in SEQ ID NO: 21. The hinge region of human IgG{3} is shown in SEQ ID NO: 22. The hinge region of human IgG{4} is shown in SEQ ID NO: 23.

[0257] In some embodiments, the hinge region according to the present disclosure comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥{94}%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity, with SEQ ID NO: 20, 21, 22 or 23.

[0258] In some embodiments, the hinge region comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity, with SEQ ID NO: 20.

[0259] In some aspects and embodiments, the hinge regions of the present disclosure include modifications to facilitate association with other hinge regions. In some embodiments, the hinge regions include modifications to facilitate heteromerization, i.e., association between non-identical hinge regions.

[0260] If the hinge region described herein includes modifications and further includes or consists of amino acid sequences within a certain threshold percentage amino acid sequence identity with respect to the reference amino acid sequence, it will be understood that any mutations with respect to the reference amino acid sequence are limited to reference sequence locations other than the modified locations.

[0261] In some embodiments and aspects, the hinge region of the present disclosure includes modifications to disrupt association with another hinge region. In some embodiments, the hinge region includes modifications to disrupt the formation of a disulfide bond between the hinge region and a hinge region provided on another polypeptide (e.g., within a polypeptide complex comprising two polypeptides, each containing a hinge region). In some embodiments, the hinge region includes modifications to replace one or more cysteine ​​residues with other amino acids (e.g., one or more cysteine ​​residues involved in the formation of a disulfide bond between the hinge region and a hinge region provided on another polypeptide).

[0262] Linker array In some embodiments, the polypeptides of this disclosure include one or more linker sequences between amino acid sequences. The linker sequences may be provided at one or both ends of one or more domains / regions of the polypeptides described herein, such as a CH3 domain, a CH2 domain, a hinge region, etc.

[0263] Linker sequences are known to those skilled in the art and are described, for example, in Chen et al., Adv. Drug Deliv. Rev. (2013) 65(10):1357-1369, which are incorporated herein by reference in their entirety. In some embodiments, the linker sequence may be a flexible linker sequence. A flexible linker sequence allows for the relative movement of the amino acid sequence linked by the linker sequence. Flexible linkers are known to those skilled in the art and some have been identified in Chen et al., Adv. Drug Deliv. Rev. (2013) 65(10):1357-1369. Flexible linker sequences often contain a high proportion of glycine and / or serine residues.

[0264] In some embodiments, the linker sequence includes at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence includes or consists of a glycine residue and a serine residue.

[0265] In some embodiments, the linker sequence has the following structure: (GxS)n or (GxS)nGm; where G = glycine, S = serine, x = 3 or 4, n = 2, 3, 4, 5 or 6, and m = 0, 1, 2 or 3. In some embodiments, x = 3, n = 3, 4, 5 or 6, and m = 0, 1, 2 or 3; or x = 4, n = 2, 3, 4 or 5, and m = 0, 1, 2 or 3. In some embodiments, x = 4 and n = 2 or 3, and m = 0. In some embodiments, x = 4 and n = 2.

[0266] In some embodiments, the linker sequence comprises one or more copies (e.g., 1, 2, 3, 4, 5, or 6) of the sequence motif G4S (e.g., in tandem). In some embodiments, the linker sequence comprises or consists of (G4S)4 or (G4S)6. In some embodiments, the linker sequence has a length of 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-30 amino acids.

[0267] additional array The polypeptides of this disclosure may include amino acid sequences to facilitate the expression, folding, transport, processing, purification, isolation, or detection of polypeptides or polypeptide complexes containing polypeptides.

[0268] In some embodiments, the polypeptides of the present disclosure include a tag to facilitate the isolation / purification of the polypeptide (and / or polypeptide complexes containing the polypeptide). Suitable tags, which may be called epitope tags, are well known in the art. For example, the polypeptide may include sequences encoding His, (e.g., (His)6), c-Myc, GST, MBP, CBP, FLAG, HA, E, or C tags. Such tags may be provided at the N-terminus and / or C-terminus of the polypeptide. Exemplarily, the polypeptide of the payload-carrying precursor complex in the experimental example includes a C tag at its C-terminus.

[0269] In some embodiments, the tag may be used to facilitate the separation of the final payload-carrying polypeptide complex from the by-product "dummy" polypeptide complex. In some embodiments, the tag may be used to deplete unreacted precursor polypeptide complexes from the reacted polypeptide exchange complex. In some embodiments, the polypeptide complex of the Disclosure (e.g., precursor polypeptide complex) comprises a polypeptide containing a given tag and a polypeptide not containing the tag. In some embodiments, both polypeptides of the polypeptide complex (e.g., by-product "dummy" polypeptide complex) contain the same tag. In some embodiments, the given tag is provided on the polypeptide of the polypeptide complex that is not included in the final payload-carrying polypeptide complex.

[0270] As an example, in the method schematically shown in Figure 1 of this disclosure, the polypeptides of the precursor "donor" and "acceptor" polypeptide complexes that are not included in the final payload-carrying polypeptide complex (i.e., the "defined labeled antibody") each contain a tag (in this example, a C tag). The C tag also provides depletion of unreacted precursor "donor" and "acceptor" polypeptide complexes (both containing polypeptides with the C tag), as well as depletion of the by-product "dummy" polypeptide complex (i.e., the "dummy dimer"), using an anti-C tag affinity column. After applying the mixture containing the "donor," "acceptor," by-product "dummy," and final payload-carrying polypeptide complex to an anti-C tag affinity column, it will be understood that the flow-through will contain only species lacking the C tag, i.e., the final payload-carrying polypeptide complex.

[0271] The polypeptides of this disclosure may include amino acid sequences to facilitate the attachment of payload and / or functional moieties. For example, the polypeptides may include sequences encoding a K tag (e.g., (Lys)6), a Q tag (e.g., Trp-Leu-Ala-Gln-Arg-Pro-His), or an Avi tag. Exemplarily, in Example 2.4 of this disclosure, a ruthenium moiety is attached to a polypeptide containing a Q tag at its C-terminus via the action of transglutaminase on K-tag-bound ruthenium. Further exemplarily, in Example 2.4 of this disclosure, the polypeptide contains an Avi tag at its N-terminus for site-specific biotin addition using the E. coli biotin ligase BirA.

[0272] The polypeptides of this disclosure may contain a signal peptide (also known as a leader sequence or signal sequence). A signal peptide typically consists of a sequence of 5 to 30 hydrophobic amino acids forming a single alpha-helix. Secreted proteins and proteins expressed on the cell surface often contain signal peptides. The signal peptide may be present at the N-terminus of the polypeptide or in the newly synthesized polypeptide. The signal peptide may provide efficient transport and secretion of the polypeptide. The signal peptide is often removed by cleavage (e.g., by signal peptidases) and therefore may not be present in the final mature form of the polypeptide secreted from the polypeptide-expressing cell.

[0273] Signal peptides are known for many proteins and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and / or can be identified / predicted using amino acid sequence analysis tools such as SignalP (Petersen et al., Nature Methods (2011) 8:785-786) or Signal-BLAST (Frank and Sippl, Bioinformatics (2008) 24:2172-2176).

[0274] In some embodiments, the polypeptides according to the Disclosure may contain one or more additional amino acids at the N-terminus and / or C-terminus of the polypeptide (e.g., 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, 5-10, 5-20, 5-30, 5-40, 5-50, 10-35, 20, 10-30, 10-40, 10-50, 20-30, 20-40, or 20-50 additional amino acids).

[0275] Specific exemplary polypeptides and polypeptide complexes In some embodiments, the polypeptides according to the present disclosure (e.g., the constituent polypeptides of a polypeptide complex) include a CH3 domain and a CH2 domain. In such embodiments, the CH2 domain can be provided upstream (i.e., at the N-terminus) of the CH3 domain in the amino acid sequence of the polypeptide. In such embodiments, the CH2 domain can be provided immediately upstream of the CH3 domain (i.e., adjacent to the CH3 domain in the amino acid sequence of the polypeptide). In some embodiments, the polypeptides according to the present disclosure include the following structure: N-terminus - [... ] - [CH2 domain] - [CH3 domain] - [... ] - C-terminus.

[0276] In some embodiments, the polypeptides according to the present disclosure (e.g., the constituent polypeptides of a polypeptide complex) include a hinge region. In such embodiments, the hinge region can be provided upstream (i.e., at the N-terminus) of the CH3 domain in the amino acid sequence of the polypeptide. In embodiments where the polypeptide includes a CH2 domain, the hinge region can be provided upstream (i.e., at the N-terminus) of the CH2 domain in the amino acid sequence of the polypeptide. In such embodiments, the hinge region can be provided immediately upstream of the CH2 domain (i.e., adjacent to the CH2 domain in the amino acid sequence of the polypeptide).

[0277] In some embodiments, the polypeptides according to the present disclosure (e.g., the constituent polypeptides of a polypeptide complex) include a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the polypeptides according to the present disclosure include the following structure: N-terminus - [... ] - [hinge region] - [CH2 domain] - [CH3 domain] - [... ] - C-terminus.

[0278] The following specific exemplary polypeptides are contemplated in connection with the present disclosure.

[0279] (A) A polypeptide comprising a CH3 domain having at least 70% amino acid sequence identity with the amino acid sequence described in any one of Sequence IDs 24-47 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0280] (B) The polypeptide described in (A), further comprising a payload portion.

[0281] (C) The polypeptide according to (A) or (B), further comprising a CH2 domain, wherein optionally the CH2 domain is N-terminus relative to the CH3 domain.

[0282] (D) The polypeptide according to (C), further comprising a hinge region, wherein the hinge region is optionally N-terminus relative to the CH2 domain.

[0283] A polypeptide described in any one of (E)(A) to (D), comprising (for example, in the amino acid sequence of the polypeptide, (i) = closest to the N-terminus and (iii) = closest to the C-terminus) the following: (i) Hinge region; (ii) CH3 domain; and (iii) A CH3 domain having at least 70% amino acid sequence identity with the amino acid sequence described in any one of Sequence IDs 24-47 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0284] The following specific exemplary polypeptide complexes are contemplated in connection with this disclosure.

[0285] Precursor polypeptide complex (1) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide includes a knob modification and a modification from one of the rows 1-49 of Table II, column A; and The CH3 domain of the second polypeptide includes a whole modification and a modification from column B in the same row of Table II, where the modification from column A was selected.

[0286] As an example, the polypeptide complex of the present disclosure according to (1) above may include: (i) a first polypeptide comprising a CH3 domain including 366W (i.e., knob modification), 370E and 439E (i.e., modification of row 1 of column A in Table II); and (ii) a second polypeptide comprising a CH3 domain including 407V, 366S, 368A (i.e., hole modification) and 356K (i.e., modification of row 1 of column B in Table II).

[0287] (2) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide includes a knob modification and a modification from column A of one of rows 1-12 in Table III; and The CH3 domain of the second polypeptide includes a whole modification and a modification from column B in the same row of Table III, where the modification from column A was selected.

[0288] (3) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide includes a knob modification and a modification from column A of one of rows 1-14 of Table IV; and The CH3 domain of the second polypeptide includes a whole modification and a modification from column B in the same row of Table IV, where the modification from column A was selected.

[0289] (4) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide includes a knob modification and a modification from column A of one of rows 1-8 of Table V; and The CH3 domain of the second polypeptide includes a whole modification and a modification from column B in the same row of Table V, where the modification from column A was selected.

[0290] (5) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide includes a knob modification and a modification from column A of one of rows 1-18 of Table VI; and The CH3 domain of the second polypeptide includes a whole modification and a modification from column B in the same row of Table VI, where the modification from column A is selected.

[0291] (6) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide includes a knob modification and a modification from column A of one of rows 1-24 of Table VII; and The CH3 domain of the second polypeptide includes a whole modification and a modification from column B in the same row of Table VII, where the modification from column A was selected.

[0292] (7) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide includes 366W and 370E; and The CH3 domain of the second polypeptide includes 407V, 366S, and 368A.

[0293] (8) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide contains 366W; and The CH3 domain of the second polypeptide includes 407V, 366S, 368A, and 357K.

[0294] (9) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide comprises 366W, 370E and 354C; and The CH3 domain of the second polypeptide includes 407V, 366S, and 368A.

[0295] (10) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide contains 366W; and The CH3 domain of the second polypeptide includes 407V, 366S, 368A, 357K, and 349C.

[0296] (11) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide includes 366W and 370E; and The CH3 domain of the second polypeptide includes 407V, 366S, 368A, and 349C.

[0297] (12) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide contains 366W and 354C; and The CH3 domain of the second polypeptide contains 407V, 366S, 368A, and 357K.

[0298] (13) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 24 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 49 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0299] (14) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 48 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 25 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0300] (15) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 26 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 49 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0301] (16) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 48 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 27 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0302] (17) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 24 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 51 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0303] (18) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 50 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 25 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0304] (19) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 28 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with Sequence ID No. 53 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0305] (20) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 52 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 29 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0306] (21) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 30 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with Sequence ID No. 53 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0307] (22) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 52 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 31 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0308] (23) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 28 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with Sequence ID No. 55 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0309] (24) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 54 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 29 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0310] (25) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 32 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 57 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0311] (26) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 56 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 33 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0312] (27) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 34 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 57 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0313] (28) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 56 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 35 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0314] (29) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 32 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with Sequence ID No. 59 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0315] (30) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 58 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 33 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0316] (31) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 36 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 61 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0317] (32) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 60 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 37 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0318] (33) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 38 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 61 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0319] (34) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 60 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with Sequence ID No. 39 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0320] (35) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 36 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 63 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0321] (36) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 62 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 37 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0322] (37) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 40 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 65 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0323] (38) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 64 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 41 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0324] (39) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 42 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 65 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0325] (40) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 64 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 43 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0326] (41) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 40 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 67 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0327] (42) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 66 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 41 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0328] (43) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 44 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 69 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0329] (44) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 68 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with Sequence ID No. 45 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0330] (45) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 46 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 69 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0331] (46) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 68 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 47 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0332] (47) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 44 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 71 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0333] (48) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 70 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with Sequence ID No. 45 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity).

[0334] (49) The polypeptide complex according to any one of (1) to (48), wherein the first polypeptide and / or the second polypeptide further comprises a payload portion.

[0335] (50) The polypeptide complex according to any one of (1) to (49), wherein the first polypeptide and / or the second polypeptide further comprises a functional moiety. Final payload-carrying polypeptide complex

[0336] (51) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide contains 366W and 370E; The CH3 domain of the second polypeptide includes 407V, 366S, 368A and 357K; and The first polypeptide or the second polypeptide further includes a payload portion.

[0337] (52) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide contains 366W, 370E, and 354C; The CH3 domain of the second polypeptide comprises 407V, 366S, 368A, 357K and 349C; and The first polypeptide or the second polypeptide further includes a payload portion.

[0338] (53) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 24 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 25 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The first polypeptide or the second polypeptide further includes a payload portion.

[0339] (54) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 26 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 27 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The first polypeptide or the second polypeptide further includes a payload portion.

[0340] (55) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 28 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 29 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The first polypeptide or the second polypeptide further includes a payload portion.

[0341] (56) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 30 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 31 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The first polypeptide or the second polypeptide further includes a payload portion.

[0342] (57) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 32 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 33 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The first polypeptide or the second polypeptide further includes a payload portion.

[0343] (58) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 34 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 35 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The first polypeptide or the second polypeptide further includes a payload portion.

[0344] (59) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 36 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 37 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The first polypeptide or the second polypeptide further includes a payload portion.

[0345] (60) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with Sequence ID No. 38 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 39 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The first polypeptide or the second polypeptide further includes a payload portion.

[0346] (61) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 40 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 41 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The first polypeptide or the second polypeptide further includes a payload portion.

[0347] (62) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 42 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 43 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The first polypeptide or the second polypeptide further includes a payload portion.

[0348] (63) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 44 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 45 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The first polypeptide or the second polypeptide further includes a payload portion.

[0349] (64) A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; Polypeptide complexes are formed by interactions between a first polypeptide and a second polypeptide, and these interactions include association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 46 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 47 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The first polypeptide or the second polypeptide further includes a payload portion.

[0350] Method for producing polypeptide complexes Aspects and embodiments of this disclosure relate to methods for producing polypeptide complexes, particularly payload-supported polypeptide complexes.

[0351] In some aspects and embodiments, the methods of the present disclosure may be referred to as methods for producing payload-supported polypeptide complexes. In some aspects and embodiments, the methods of the present disclosure may be referred to as methods for providing polypeptides having a payload (e.g., labeling polypeptides with a payload).

[0352] This method generally involves contacting a first polypeptide complex, comprising a first polypeptide and a second polypeptide, with a second polypeptide complex, comprising a third polypeptide and a fourth polypeptide, to form a third polypeptide complex, comprising a first polypeptide and a fourth polypeptide, and / or a fourth polypeptide complex, comprising a second polypeptide and a third polypeptide.

[0353] The first polypeptide complex and the second polypeptide complex are brought into contact with each other under conditions suitable for the exchange of their constituent polypeptides and the formation of a third polypeptide complex and / or a fourth polypeptide complex. In some embodiments, the method is carried out essentially as described in Example 1.3 of this disclosure.

[0354] This method may involve appropriately mixing equimolar amounts of a first polypeptide complex and a second polypeptide complex. It will be understood that the first and second polypeptide complexes are provided at concentrations that allow for efficient exchange of their constituent polypeptides. In some embodiments, equimolar amounts of the first and second polypeptide complexes are mixed at concentrations of 0.1 to 10 mg / ml, for example, 0.5 to 2 mg / ml (for example, about 1 mg / ml).

[0355] The first polypeptide complex and the second polypeptide complex can be brought into contact with each other in a suitable buffer. As used herein, “buffer” refers to a buffer solution that is resistant to changes in pH due to the action of its acid-base conjugate components. In some embodiments, the buffer is or contains phosphate-buffered saline (PBS) at pH 7.4.

[0356] In some embodiments (for example, embodiments in which the first polypeptide complex and / or the second polypeptide complex are formed by interactions between constituent polypeptides including one or more interchain disulfide bonds, e.g., between cysteine ​​residues in the hinge region of the polypeptides), the method may include treating the first polypeptide complex and / or the second polypeptide complex with a reducing agent. The reducing agent can break the disulfide bonds of the treated polypeptide complex. In some embodiments, the reducing agent is tris(2-carboxyethyl)phosphine (TCEP). In some embodiments, the reducing agent is provided to the mixture of the first polypeptide complex and the second polypeptide complex in a molar excess, e.g., 2 to 50 molar excess, e.g., 10 to 30 molar excess or 15 to 25 molar excess (e.g., about 20 molar excess).

[0357] In some embodiments (for example, embodiments in which the first polypeptide complex and / or the second polypeptide complex are formed by interactions between constituent polypeptides that do not contain interchain disulfide bonds), the method may not include treating the first polypeptide complex and / or the second polypeptide complex with a reducing agent.

[0358] A mixture containing the first polypeptide complex and the second polypeptide complex is incubated at a temperature that provides a favorable kinetics for the chain exchange of their constituent polypeptides. In some embodiments, the mixture containing the first polypeptide complex and the second polypeptide complex is incubated at a temperature of 4°C to 60°C, for example, 15°C to 40°C or 25°C to 40°C (e.g., about 37°C).

[0359] A mixture containing the first polypeptide complex and the second polypeptide complex is incubated for a period of time sufficient for chain exchange of their constituent polypeptides to occur. In some embodiments, the mixture containing the first polypeptide complex and the second polypeptide complex is incubated for 30 minutes to 12 hours, for example, 1 to 6 hours or 2 to 4 hours (for example, about 3 hours).

[0360] The incubation of the mixture containing the first polypeptide complex and the second polypeptide complex may be carried out with stirring to facilitate mixing. In some embodiments, the mixture containing the first polypeptide complex and the second polypeptide complex is incubated with stirring at 100 rpm to 600 rpm, for example, 150 rpm to 500 rpm or 200 rpm to 400 rpm (for example, about 300 rpm).

[0361] The method of the present disclosure includes recovering a third polypeptide complex and / or a fourth polypeptide complex after incubation of a first polypeptide complex and a second polypeptide complex. In some embodiments, “recovering” the polypeptide complex includes separating / isolating / purifying and / or recovering the polypeptide complex.

[0362] In some embodiments, the methods of the present disclosure include separating / isolating / purifying the polypeptide complex. For example, after incubation of a first polypeptide complex and a second polypeptide complex for polypeptide chain exchange, the reaction product may be isolated / purified / depleted. In some embodiments, the methods of the present disclosure include separating / isolating / purifying the polypeptide complex by affinity chromatography. Affinity chromatography is well known in the art, and its principles are described, for example, in Rodriguez et al., J.Chromatogr.B.Analyt.Technol.Biomed.Life Sci. (2020) 1157:122332, which is incorporated herein by reference in its entirety.

[0363] In some embodiments, the method of the present disclosure includes contacting a mixture comprising first, second, third, and fourth polypeptide complexes with an agent to facilitate the separation of one or more polypeptide complexes from the mixture.

[0364] In embodiments in which the constituent polypeptides of the first polypeptide complex and / or the second polypeptide complex contain a suitable tag, the third polypeptide complex and / or the fourth polypeptide complex may be isolated / purified / depleted using an affinity purifying agent for the polypeptide complex containing the tag. Alternatively, or further, if the constituent polypeptides of the first polypeptide complex and / or the second polypeptide complex contain a suitable tag, the unreacted precursor polypeptide complex may be isolated / purified / depleted from the third polypeptide complex and / or the fourth polypeptide complex using an affinity purifying agent for such precursor polypeptide complex containing the tag.

[0365] As an example, in the method schematically shown in Figure 1 of this disclosure, the polypeptides of the first and second polypeptide complexes (i.e., precursor "donor" and "acceptor" polypeptide complexes) that are not included in the final payload-carrying polypeptide complex (i.e., the "defined labeled antibody") each contain a tag (in this example, a C tag). The C tag also provides depletion of unreacted precursor "donor" and "acceptor" polypeptide complexes (both containing polypeptides with the C tag) using an anti-C tag affinity column, as well as depletion of the by-product "dummy" polypeptide complex (i.e., the "dummy dimer"). After applying the mixture containing the "donor," "acceptor," by-product "dummy," and final payload-carrying polypeptide complex to an anti-C tag affinity column, the flow-through contains only species lacking the C tag, i.e., the final payload-carrying polypeptide complex.

[0366] In the example in the previous paragraph, the agent for affinity purification of the polypeptide complex, including the tag (i.e., the anti-C tag affinity column), is used to isolate / purify the final payload-carrying polypeptide complex by depleting the unreacted precursor "donor" and "acceptor" polypeptide complexes, as well as the by-product "dummy" polypeptide complex. However, in an alternative embodiment, the agent may be used to isolate / purify the final payload-carrying polypeptide complex by positive selection.

[0367] In some embodiments, an agent for facilitating the separation of one or more polypeptide complexes from a mixture comprising the first, second, third, and fourth polypeptide complexes comprises a portion for binding to a tag (i.e., a tag contained in one or more polypeptides of the polypeptide complex). In some embodiments, the tag may be, for example, an epitope tag as described herein. In some embodiments, the portion for binding to the tag may be or contain an antibody specific to the tag, or an antigen-binding fragment / derivative thereof. In some embodiments, the portion for binding to the tag may be provided on a solid substrate, such as beads (e.g., agarose beads). In some embodiments, beads containing the portion for binding to the tag may be provided in a column.

[0368] The separation / isolation / purification of one or more polypeptide complexes according to this disclosure may be alternatively or additionally performed using further techniques for separating / isolating / purifying polypeptide complexes. Such techniques include, for example, size exclusion chromatography, capillary electrophoresis, and ion exchange chromatography. In some embodiments, the method of this disclosure includes separating / isolating / purifying polypeptide complexes by size exclusion chromatography and / or capillary electrophoresis.

[0369] Specific exemplary methods for producing payload-supported polypeptide complexes The following specific exemplary methods for producing polypeptide payload-supported polypeptide complexes according to this disclosure are intended: (1) A method for producing a polypeptide complex, below: (i) A first polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; The first polypeptide complex is formed by an interaction between the first polypeptide and the second polypeptide, and this interaction involves the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide contains 366W and 370E; The CH3 domain of the second polypeptide is part of the first polypeptide complex, containing 407V, 366S, and 368A; (ii) A second polypeptide complex comprising a third polypeptide containing a CH3 domain and a fourth polypeptide containing a CH3 domain; The second polypeptide complex is formed by an interaction between the third polypeptide and the fourth polypeptide, the interaction involving the association between the CH3 domain of the third polypeptide and the CH3 domain of the fourth polypeptide; The CH3 domain of the third polypeptide contains 366W; The CH3 domain of the fourth polypeptide, containing 407V, 366S, 368A, and 357K, is incubated with the second polypeptide complex; To form a third polypeptide complex comprising the first polypeptide and the fourth polypeptide (i.e., the polypeptide complex described in (51) above), and / or a fourth polypeptide complex comprising the second polypeptide and the third polypeptide; and The process includes recovering the third polypeptide complex and / or the fourth polypeptide complex; The first and / or second polypeptide of the first polypeptide complex or the first and / or second polypeptide of the second polypeptide complex further comprises a payload portion.

[0370] (2) A method for producing a polypeptide complex, below: (i) A first polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; The first polypeptide complex is formed by an interaction between the first polypeptide and the second polypeptide, and this interaction involves the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide comprises 366W, 370E and 354C; and The CH3 domain of the second polypeptide is part of the first polypeptide complex, containing 407V, 366S, and 368A; (ii) A second polypeptide complex comprising a third polypeptide containing a CH3 domain and a fourth polypeptide containing a CH3 domain; The second polypeptide complex is formed by an interaction between the third polypeptide and the fourth polypeptide, the interaction involving the association between the CH3 domain of the third polypeptide and the CH3 domain of the fourth polypeptide; The CH3 domain of the third polypeptide contains 366W; and The CH3 domain of the fourth polypeptide, containing 407V, 366S, 368A, 357K, and 349C, is incubated with the second polypeptide complex; To form a third polypeptide complex comprising the first polypeptide and the fourth polypeptide (i.e., the polypeptide complex described in (52) above), and / or a fourth polypeptide complex comprising the second polypeptide and the third polypeptide; and The process includes recovering the third polypeptide complex and / or the fourth polypeptide complex; The first and / or second polypeptide of the first polypeptide complex or the first and / or second polypeptide of the second polypeptide complex further comprises a payload portion.

[0371] (3) A method for producing a polypeptide complex, below: (i) A first polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; The first polypeptide complex is formed by an interaction between the first polypeptide and the second polypeptide, and this interaction involves the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 24 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 49 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity) in the first polypeptide complex; (ii) A second polypeptide complex comprising a third polypeptide containing a CH3 domain and a fourth polypeptide containing a CH3 domain; The second polypeptide complex is formed by an interaction between the third polypeptide and the fourth polypeptide, the interaction involving the association between the CH3 domain of the third polypeptide and the CH3 domain of the fourth polypeptide; The CH3 domain of the third polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 48 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the fourth polypeptide is incubated with the second polypeptide complex having at least 70% amino acid sequence identity with SEQ ID NO: 25 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); To form a third polypeptide complex comprising the first polypeptide and the fourth polypeptide (i.e., the polypeptide complex described in (53) above), and / or a fourth polypeptide complex comprising the second polypeptide and the third polypeptide; and The process includes recovering the third polypeptide complex and / or the fourth polypeptide complex; The first and / or second polypeptide of the first polypeptide complex or the first and / or second polypeptide of the second polypeptide complex further comprises a payload portion.

[0372] (4) A method for producing a polypeptide complex, below: (i) A first polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; The first polypeptide complex is formed by an interaction between the first polypeptide and the second polypeptide, and this interaction involves the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 26 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 49 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity) in the first polypeptide complex; (ii) A second polypeptide complex comprising a third polypeptide containing a CH3 domain and a fourth polypeptide containing a CH3 domain; The second polypeptide complex is formed by an interaction between the third polypeptide and the fourth polypeptide, the interaction involving the association between the CH3 domain of the third polypeptide and the CH3 domain of the fourth polypeptide; The CH3 domain of the third polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 48 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the fourth polypeptide is incubated with the second polypeptide complex having at least 70% amino acid sequence identity with SEQ ID NO: 27 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); To form a third polypeptide complex comprising the first polypeptide and the fourth polypeptide (i.e., the polypeptide complex described in (54) above), and / or a fourth polypeptide complex comprising the second polypeptide and the third polypeptide; and The process includes recovering the third polypeptide complex and / or the fourth polypeptide complex; The first and / or second polypeptide of the first polypeptide complex or the first and / or second polypeptide of the second polypeptide complex further comprises a payload portion.

[0373] (5) A method for producing a polypeptide complex, below: (i) A first polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; The first polypeptide complex is formed by an interaction between the first polypeptide and the second polypeptide, and this interaction involves the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 28 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 53 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity) in the first polypeptide complex; (ii) A second polypeptide complex comprising a third polypeptide containing a CH3 domain and a fourth polypeptide containing a CH3 domain; The second polypeptide complex is formed by an interaction between the third polypeptide and the fourth polypeptide, the interaction involving the association between the CH3 domain of the third polypeptide and the CH3 domain of the fourth polypeptide; The CH3 domain of the third polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 52 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the fourth polypeptide is incubated with the second polypeptide complex having at least 70% amino acid sequence identity with SEQ ID NO: 29 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); To form a third polypeptide complex comprising the first polypeptide and the fourth polypeptide (i.e., the polypeptide complex described in (55) above), and / or a fourth polypeptide complex comprising the second polypeptide and the third polypeptide; and The process includes recovering the third polypeptide complex and / or the fourth polypeptide complex; The first and / or second polypeptide of the first polypeptide complex or the first and / or second polypeptide of the second polypeptide complex further comprises a payload portion.

[0374] (5) A method for producing a polypeptide complex, below: (i) A first polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; The first polypeptide complex is formed by an interaction between the first polypeptide and the second polypeptide, and this interaction involves the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 30 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 53 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity) in the first polypeptide complex; (ii) A second polypeptide complex comprising a third polypeptide containing a CH3 domain and a fourth polypeptide containing a CH3 domain; The second polypeptide complex is formed by an interaction between the third polypeptide and the fourth polypeptide, the interaction involving the association between the CH3 domain of the third polypeptide and the CH3 domain of the fourth polypeptide; The CH3 domain of the third polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 52 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the fourth polypeptide is incubated with the second polypeptide complex having at least 70% amino acid sequence identity with SEQ ID NO: 31 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); To form a third polypeptide complex comprising the first polypeptide and the fourth polypeptide (i.e., the polypeptide complex described in (56) above), and / or a fourth polypeptide complex comprising the second polypeptide and the third polypeptide; and The process includes recovering the third polypeptide complex and / or the fourth polypeptide complex; The first and / or second polypeptide of the first polypeptide complex or the first and / or second polypeptide of the second polypeptide complex further comprises a payload portion.

[0375] (7) A method for producing a polypeptide complex, below: (i) A first polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; The first polypeptide complex is formed by an interaction between the first polypeptide and the second polypeptide, and this interaction involves the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 32 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 57 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity) of the first polypeptide complex; (ii) A second polypeptide complex comprising a third polypeptide containing a CH3 domain and a fourth polypeptide containing a CH3 domain; The second polypeptide complex is formed by an interaction between the third polypeptide and the fourth polypeptide, the interaction involving the association between the CH3 domain of the third polypeptide and the CH3 domain of the fourth polypeptide; The CH3 domain of the third polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 56 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the fourth polypeptide is incubated with the second polypeptide complex having at least 70% amino acid sequence identity with SEQ ID NO: 33 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); To form a third polypeptide complex comprising the first polypeptide and the fourth polypeptide (i.e., the polypeptide complex described in (57) above), and / or a fourth polypeptide complex comprising the second polypeptide and the third polypeptide; and The process includes recovering the third polypeptide complex and / or the fourth polypeptide complex; The first and / or second polypeptide of the first polypeptide complex or the first and / or second polypeptide of the second polypeptide complex further comprises a payload portion.

[0376] (8) A method for producing a polypeptide complex, below: (i) A first polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; The first polypeptide complex is formed by an interaction between the first polypeptide and the second polypeptide, and this interaction involves the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 34 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 57 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity) of the first polypeptide complex; (ii) A second polypeptide complex comprising a third polypeptide containing a CH3 domain and a fourth polypeptide containing a CH3 domain; The second polypeptide complex is formed by an interaction between the third polypeptide and the fourth polypeptide, the interaction involving the association between the CH3 domain of the third polypeptide and the CH3 domain of the fourth polypeptide; The CH3 domain of the third polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 56 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the fourth polypeptide is incubated with the second polypeptide complex having at least 70% amino acid sequence identity with SEQ ID NO: 35 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); To form a third polypeptide complex comprising the first polypeptide and the fourth polypeptide (i.e., the polypeptide complex described in (58) above), and / or a fourth polypeptide complex comprising the second polypeptide and the third polypeptide; and The process includes recovering the third polypeptide complex and / or the fourth polypeptide complex; The first and / or second polypeptide of the first polypeptide complex or the first and / or second polypeptide of the second polypeptide complex further comprises a payload portion.

[0377] (9) A method for producing a polypeptide complex, below: (i) A first polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; The first polypeptide complex is formed by an interaction between the first polypeptide and the second polypeptide, and this interaction involves the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 36 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 61 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity) in the first polypeptide complex; (ii) A second polypeptide complex comprising a third polypeptide containing a CH3 domain and a fourth polypeptide containing a CH3 domain; The second polypeptide complex is formed by an interaction between the third polypeptide and the fourth polypeptide, the interaction involving the association between the CH3 domain of the third polypeptide and the CH3 domain of the fourth polypeptide; The CH3 domain of the third polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 60 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the fourth polypeptide is incubated with the second polypeptide complex having at least 70% amino acid sequence identity with SEQ ID NO: 37 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); To form a third polypeptide complex comprising the first polypeptide and the fourth polypeptide (i.e., the polypeptide complex described in (59) above), and / or a fourth polypeptide complex comprising the second polypeptide and the third polypeptide; and The process includes recovering the third polypeptide complex and / or the fourth polypeptide complex; The first and / or second polypeptide of the first polypeptide complex or the first and / or second polypeptide of the second polypeptide complex further comprises a payload portion.

[0378] (10) A method for producing a polypeptide complex, below: (i) A first polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; The first polypeptide complex is formed by an interaction between the first polypeptide and the second polypeptide, and this interaction involves the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 38 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 61 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity) in the first polypeptide complex; (ii) A second polypeptide complex comprising a third polypeptide containing a CH3 domain and a fourth polypeptide containing a CH3 domain; The second polypeptide complex is formed by an interaction between the third polypeptide and the fourth polypeptide, the interaction involving the association between the CH3 domain of the third polypeptide and the CH3 domain of the fourth polypeptide; The CH3 domain of the third polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 60 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the fourth polypeptide is incubated with the second polypeptide complex having at least 70% amino acid sequence identity with SEQ ID NO: 39 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); To form a third polypeptide complex comprising the first polypeptide and the fourth polypeptide (i.e., the polypeptide complex described in (60) above), and / or a fourth polypeptide complex comprising the second polypeptide and the third polypeptide; and The process includes recovering the third polypeptide complex and / or the fourth polypeptide complex; The first and / or second polypeptide of the first polypeptide complex or the first and / or second polypeptide of the second polypeptide complex further comprises a payload portion.

[0379] (11) A method for producing a polypeptide complex, below: (i) A first polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; The first polypeptide complex is formed by an interaction between the first polypeptide and the second polypeptide, and this interaction involves the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 40 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 65 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity) in the first polypeptide complex; (ii) A second polypeptide complex comprising a third polypeptide containing a CH3 domain and a fourth polypeptide containing a CH3 domain; The second polypeptide complex is formed by an interaction between the third polypeptide and the fourth polypeptide, the interaction involving the association between the CH3 domain of the third polypeptide and the CH3 domain of the fourth polypeptide; The CH3 domain of the third polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 64 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the fourth polypeptide is incubated with the second polypeptide complex having at least 70% amino acid sequence identity with SEQ ID NO: 41 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); To form a third polypeptide complex comprising the first polypeptide and the fourth polypeptide (i.e., the polypeptide complex described in (61) above), and / or a fourth polypeptide complex comprising the second polypeptide and the third polypeptide; and The process includes recovering the third polypeptide complex and / or the fourth polypeptide complex; The first and / or second polypeptide of the first polypeptide complex or the first and / or second polypeptide of the second polypeptide complex further comprises a payload portion.

[0380] (12) A method for producing a polypeptide complex, below: (i) A first polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; The first polypeptide complex is formed by an interaction between the first polypeptide and the second polypeptide, and this interaction involves the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 42 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 65 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity) in the first polypeptide complex; (ii) A second polypeptide complex comprising a third polypeptide containing a CH3 domain and a fourth polypeptide containing a CH3 domain; The second polypeptide complex is formed by an interaction between the third polypeptide and the fourth polypeptide, the interaction involving the association between the CH3 domain of the third polypeptide and the CH3 domain of the fourth polypeptide; The CH3 domain of the third polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 64 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the fourth polypeptide is incubated with the second polypeptide complex having at least 70% amino acid sequence identity with SEQ ID NO: 43 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); To form a third polypeptide complex comprising the first polypeptide and the fourth polypeptide (i.e., the polypeptide complex described in (62) above), and / or a fourth polypeptide complex comprising the second polypeptide and the third polypeptide; and The process includes recovering the third polypeptide complex and / or the fourth polypeptide complex; The first and / or second polypeptide of the first polypeptide complex or the first and / or second polypeptide of the second polypeptide complex further comprises a payload portion.

[0381] (13) A method for producing a polypeptide complex, below: (i) A first polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; The first polypeptide complex is formed by an interaction between the first polypeptide and the second polypeptide, and this interaction involves the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 44 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 69 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity) in the first polypeptide complex; (ii) A second polypeptide complex comprising a third polypeptide containing a CH3 domain and a fourth polypeptide containing a CH3 domain; The second polypeptide complex is formed by an interaction between the third polypeptide and the fourth polypeptide, the interaction involving the association between the CH3 domain of the third polypeptide and the CH3 domain of the fourth polypeptide; The CH3 domain of the third polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 68 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the fourth polypeptide is incubated with the second polypeptide complex having at least 70% amino acid sequence identity with SEQ ID NO: 45 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); To form a third polypeptide complex comprising the first polypeptide and the fourth polypeptide (i.e., the polypeptide complex described in (63) above), and / or a fourth polypeptide complex comprising the second polypeptide and the third polypeptide; and The process includes recovering the third polypeptide complex and / or the fourth polypeptide complex; The first and / or second polypeptide of the first polypeptide complex or the first and / or second polypeptide of the second polypeptide complex further comprises a payload portion.

[0382] (14) A method for producing a polypeptide complex, below: (i) A first polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain; The first polypeptide complex is formed by an interaction between the first polypeptide and the second polypeptide, and this interaction involves the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide; The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 46 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 69 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity) in the first polypeptide complex; (ii) A second polypeptide complex comprising a third polypeptide containing a CH3 domain and a fourth polypeptide containing a CH3 domain; The second polypeptide complex is formed by an interaction between the third polypeptide and the fourth polypeptide, the interaction involving the association between the CH3 domain of the third polypeptide and the CH3 domain of the fourth polypeptide; The CH3 domain of the third polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 68 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); and The CH3 domain of the fourth polypeptide is incubated with the second polypeptide complex having at least 70% amino acid sequence identity with SEQ ID NO: 47 (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity); To form a third polypeptide complex comprising the first polypeptide and the fourth polypeptide (i.e., the polypeptide complex described in (64) above), and / or a fourth polypeptide complex comprising the second polypeptide and the third polypeptide; and The process includes recovering the third polypeptide complex and / or the fourth polypeptide complex; The first and / or second polypeptide of the first polypeptide complex or the first and / or second polypeptide of the second polypeptide complex further comprises a payload portion.

[0383] Characteristics of the final payload-carrying polypeptide complex The present disclosure provides a method for producing the final payload-supported polypeptide complex, and the final payload-supported polypeptide complex produced by this method offers advantageous properties.

[0384] The method disclosed herein provides for the production of payload-supported polypeptide complexes by precisely controlling the amount of the payload portion in the final molecule.

[0385] For example, if the payload portion is attached to the polypeptide of the donor precursor complex via nonspecific conjugation (e.g., an NHS-containing payload portion to a lysine residue), the method of the present disclosure provides the production of a final payload-supported polypeptide complex with high consistency in the number of payload portions compared to conventional methods for labeling the polypeptide and its complex with the payload portion. Exemplarily, Example 2.2 of the present disclosure illustrates that different antibodies (i.e., containing non-identical antigen-binding domains) can be produced from a single pool of “donor” precursor complexes having very similar payload portion-to-antigen-binding portion ratios (e.g., drug-antibody ratio; DAR) using the method of the present disclosure.

[0386] Because the precise number of payload portions per molecule can be determined, the final payload-supported polypeptide complexes of this disclosure are particularly well suited to applications involving relative and / or absolute quantification.

[0387] For the same reason, the final payload-supported polypeptide complexes of this disclosure are also particularly well suited for use in comparing different functional domains. For example, as shown in Example 2.2, molecules containing different antigen-binding moieties contain the same amount of payload moiety, so their functional properties (in this example, internal migration efficiency) can be directly compared.

[0388] The method of this disclosure also provides control over where on the molecule the payload portion is provided in a molecule labeled by nonspecific conjugation. For example, as shown in Example 2.2, the nonspecific conjugation step is performed independently of the functional portion (in this example, the antigen-binding domain of the final molecule), so that the functional portion remains unchanged.

[0389] It will also be understood that the methods disclosed herein provide efficient production of a wide variety of different species, including different functional portions and different payload portions. This modular approach allows a “donor” polypeptide complex to provide a polypeptide containing essentially any payload portion to a polypeptide of an “acceptor” polypeptide complex containing essentially any functional portion.

[0390] nucleic acids and vectors This disclosure provides nucleic acids or a number of nucleic acids that encode one or more polypeptides of the (precursor) polypeptide complex according to this disclosure. In some embodiments, the nucleic acids include or consist of DNA and / or RNA.

[0391] This disclosure also provides a vector or multiple vectors comprising nucleic acids or multiple nucleic acids as a result of this disclosure.

[0392] The nucleic acids and vectors provided herein may be provided in purified or isolated forms, i.e., from other nucleic acids or naturally occurring biomaterials.

[0393] Nucleotide sequences may be contained in vectors, such as expression vectors. As used herein, “vector” is a nucleic acid molecule used as a vehicle for transferring exogenous nucleic acids into cells. A vector may be a vector for the expression of nucleic acids in cells. Such a vector may include a promoter sequence operably ligated to a nucleotide sequence encoding the sequence to be expressed. A vector may also include a stop codon and an expression enhancer. Peptides or polypeptides may be expressed from the vector according to this disclosure using any suitable vector, promoter, enhancer, and stop codon known in the art.

[0394] The term "operably ligated" may include situations where a selected nucleic acid sequence and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked in such a way that the expression of the nucleic acid sequence is influenced by or controlled by the regulatory sequence (thereby forming an expression cassette). Therefore, if the regulatory sequence can transcribe the nucleic acid sequence, then the regulatory sequence is operably ligated to the selected nucleic acid sequence. The resulting transcript can then be translated into the desired peptide / polypeptide.

[0395] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., gamma retrovirus vectors (e.g., mouse leukemia virus (MLV)-derived vectors), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, and herpesvirus vectors)), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes).

[0396] In some embodiments, the vector may be a eukaryotic vector, for example, a vector containing elements necessary for protein expression from the vector in eukaryotic cells. In some embodiments, the vector may be a mammalian vector containing, for example, a cytomegalovirus (CMV) or SV40 promoter that drives protein expression.

[0397] The constituent polypeptides of the polypeptide complex according to this disclosure may be encoded by different nucleic acids from among multiple nucleic acids, or by different vectors from among multiple vectors.

[0398] Cells containing / expressing polypeptide / precursor polypeptide complexes This disclosure also provides cells containing or expressing polypeptides or (precursor) polypeptide complexes according to this disclosure. Cells containing or expressing nucleic acids or vectors according to this disclosure are also provided.

[0399] The cells may be eukaryotic cells, such as mammalian cells. Mammals may be primates (rhesus monkeys, crab-eating macaques, non-human primates, or humans) or non-human mammals (e.g., rabbits, guinea pigs, rats, mice or other rodents (including any animals of the order Rodentia), cats, dogs, pigs, sheep, goats, cattle (including cows, such as dairy cows, or any animals of the order Artiodactyla), horses (including any animals of the order Perissodactyla), donkeys, and non-human primates).

[0400] In some embodiments, the cells are or are derived from cell types commonly used for the expression of polypeptides for therapeutic use, for example, in humans. Exemplary cells are described, for example, Kunert and Reinhart, Appl. Microbiol. Biotechnol. (2016) 100:3451-3461 (the whole of which is incorporated herein by reference), and include, for example, CHO, HEK 293, PER.C6, NS0, and BHK cells.

[0401] The Disclosure also provides a method for producing cells comprising nucleic acids or vectors according to the Disclosure, the method comprising introducing the nucleic acids or vectors according to the Disclosure into cells. In some embodiments, introducing isolated nucleic acids or vectors according to the Disclosure into cells includes transformation, transfection, electroporation, or transduction (e.g., retroviral transduction).

[0402] The Disclosure also provides a method for producing cells expressing / containing a polypeptide or polypeptide complex according to the Disclosure, which includes introducing a nucleic acid or vector according to the Disclosure into cells. In some embodiments, the Method further includes culturing cells under conditions suitable for nucleic acid / vector expression by cells. In some embodiments, the Method is performed in vitro.

[0403] This disclosure also provides cells that can be obtained or obtained by the methods relating to this disclosure.

[0404] Production of polypeptide and precursor polypeptide complexes The polypeptides and (precursor) polypeptide complexes according to this disclosure can be prepared according to polypeptide production methods known to those skilled in the art.

[0405] Polypeptides can be prepared by chemical synthesis, for example, liquid or solid-phase synthesis. For example, peptides / polypeptides can be synthesized using the methods described, for example, Chandrudu et al., Molecules (2013), 18:4373-4388 (the entire work is incorporated herein by reference).

[0406] Alternatively, polypeptides and polypeptide complexes can be produced by recombinant expression. Suitable molecular biology techniques for recombinant polypeptide production are well known in the art and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Press (2012), and Nat Methods. (2008); 5(2): 135-146, both of which are incorporated herein by reference in their entirety.

[0407] Any cell suitable for polypeptide expression may be used for recombinant production according to this disclosure. The cell may be a prokaryote or a eukaryote. In some embodiments, the cell may be a prokaryotic cell, such as an archaeal or bacterial cell. In some embodiments, the bacterium may be a Gram-negative bacterium, such as a bacterium of the Enterobacteriaceae family, such as Escherichia coli. In some embodiments, the cell may be a eukaryotic cell, such as a yeast cell, plant cell, insect cell, or mammalian cell, such as the cells described above herein.

[0408] In some cases, certain prokaryotic cells do not allow the same folding or post-translational modifications as eukaryotic cells, and therefore the cells are not prokaryotic. Furthermore, very high expression levels are possible in eukaryotes, and proteins can be purified more easily from eukaryotes using appropriate tags. Specific plasmids that enhance the secretion of proteins into the culture medium may also be available.

[0409] In some embodiments, polypeptides may be prepared by cell-free protein synthesis (CFPS) according to a system described, for example, in Zemella et al. Chembiochem (2015) 16(17):2420-2431, which is incorporated entirely herein by reference.

[0410] Production may involve culturing or fermenting eukaryotic cells modified to express the polypeptide of interest. Culturing or fermentation may be carried out in a bioreactor with adequate supply of nutrients, air / oxygen, and / or growth factors. Secreted proteins can be collected by fractionating the culture medium / fermentation broth from the cells, extracting the protein content, and isolating the secreted polypeptides by separating individual proteins. Culturing, fermentation, and isolation techniques are well known to those skilled in the art and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition; incorporated herein by reference above).

[0411] A bioreactor comprises one or more vessels in which cells can be cultured. Culture within the bioreactor may be carried out continuously, with a continuous flow of reactants into the reactor and a continuous flow of cultured cells from the reactor. Alternatively, culture may be carried out in batches. The bioreactor monitors and controls environmental conditions such as pH, oxygen, inflow and outflow into the vessels, and agitation within the vessels to provide optimal conditions for the cells being cultured.

[0412] After culturing cells expressing a polypeptide, the polypeptide can be isolated or purified (e.g., from the cell culture supernatant). Any suitable method can be used to isolate / purify the polypeptide of interest produced by expression from cells in culture.

[0413] To isolate expressed polypeptides, it may be necessary to separate cells from the nutrient medium. If the polypeptide of interest is secreted from the cells, the cells can be separated from the culture medium containing the secreted polypeptide of interest by centrifugation. If the polypeptide of interest is recovered within the cells, protein isolation may involve centrifugation to separate the cells from the cell culture medium, processing of the cell pellet with lysis buffer, and cell disruption by means of, for example, sonication, rapid freeze-thaw, or osmotic lysis.

[0414] Next, it may be desirable to isolate the target polypeptide from the supernatant or culture medium, which may contain other protein and non-protein components. A common approach to separating protein components from the supernatant or culture medium is by precipitation. Proteins with different solubility are precipitated with different concentrations of precipitating agents, such as ammonium sulfate. For example, low concentrations of precipitating agents extract water-soluble proteins. Therefore, proteins with different solubility can be distinguished by adding increasing concentrations of precipitating agents. Subsequently, ammonium sulfate can be removed from the separated proteins using dialysis.

[0415] Other methods for distinguishing different proteins, such as ion-exchange chromatography and size chromatography, are known in the art. These may be used as an alternative to precipitation or performed following precipitation.

[0416] Once the target polypeptide is isolated from the culture, it may be desirable or necessary to concentrate the polypeptide. Several methods for concentrating proteins are known in the art, such as ultrafiltration or lyophilization.

[0417] composition This disclosure also provides compositions comprising polypeptides, polypeptide complexes, nucleic acids, expression vectors, and cells as described herein.

[0418] The polypeptides, polypeptide complexes, nucleic acids, expression vectors, and cells described herein may be formulated as pharmaceutical compositions or pharmaceuticals for use in therapeutic and / or prophylactic methods, and may include pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. The polypeptides, polypeptide complexes, nucleic acids, expression vectors, and cells described herein may be formulated for use in diagnostic and / or prognostic applications.

[0419] The compositions disclosed herein include one or more pharmaceutically acceptable carriers (e.g., liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g., starch, cellulose, cellulose derivatives, polyols, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methylparaben, propylparaben), and antioxidants. It may contain (for example, vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (for example, magnesium stearate, talc, silica, stearic acid, vegetable stearin), binders (for example, sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilizers, solubilizers, surfactants (for example, wetting agents), masking agents or colorants (for example, titanium dioxide).

[0420] As used herein, the term “pharmaceutically acceptable” means that, within the bounds of sound medical judgment, a compound, component, material, composition, dosage form, etc., is suitable for use in contact with the tissue of the subject of concern (e.g., a human subject) without excessive toxicity, irritation, allergic reaction, or other problems or complications, and is commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, binder, stabilizer, solubilizer, surfactant, masking agent, colorant, flavoring agent, or sweetener of a composition according to this disclosure must also be “acceptable” in the sense that it is compatible with the other components of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilizers, solubilizers, surfactants, masking agents, colorants, flavoring agents, or sweeteners can be found in standard pharmaceutical texts, such as Remington's "The Science and Practice of Pharmacy" (Ed. A. Adejare), 23rd Edition (2020), Academic Press.

[0421] The composition may be formulated for local, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral, or transdermal administration routes. In some embodiments, the pharmaceutical composition / pharmaceutical may be formulated for administration by injection or infusion, or by ingestion.

[0422] Suitable formulations may contain the relevant articles in a sterile or isotonic medium. Pharmaceuticals and pharmaceutical compositions may be formulated in fluid forms, including gels. Fluid formulations may be formulated for administration by injection or infusion (e.g., via a catheter) into a selected area of ​​the human or animal body.

[0423] In some embodiments, the composition is formulated for injection or infusion into, for example, a blood vessel or tissue / organ of interest.

[0424] This disclosure also provides methods for producing pharmaceutically useful compositions, such production methods may comprise one or more steps selected from: producing polypeptide / polypeptide complexes, nucleic acids, expression vectors or cells described herein; isolating polypeptide / polypeptide complexes, nucleic acids, expression vectors or cells described herein; and / or mixing polypeptide / polypeptide complexes, nucleic acids, expression vectors or cells described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

[0425] Purpose The final payload-supported polypeptide complex of this disclosure (and articles containing it) may be used in any suitable application selected according to the identity of the payload portion and / or functional portion.

[0426] In particular, the use of the polypeptide complexes of this disclosure is intended for therapeutic, preventive, diagnostic, and prognostic applications.

[0427] For example, if the functional portion is or contains a target antigen-binding peptide / polypeptide and the payload portion is a detectable portion, the final payload-carrying polypeptide complex may be used in methods that include detecting and / or quantifying the relevant target antigen, such as diagnostic and / or prognostic diagnostic methods (e.g., when the target antigen is a disease-associated antigen).

[0428] For example, if the functional portion is or contains a target-binding peptide / polypeptide and the payload portion is a drug portion, the final payload-carrying polypeptide complex may be used in methods that involve depleting cells expressing the relevant target antigen (e.g., on the cell surface), such as therapeutic and / or preventive methods (e.g., when the target antigen is a disease-associated antigen).

[0429] kit This disclosure also provides a kit of parts. In some embodiments, the kit may include at least one container having a predetermined amount of polypeptides, polypeptide complexes, nucleic acids, vectors, cells, or compositions as described herein.

[0430] The kit may include instructions for use, for example, in the form of an instruction booklet or leaflet. The instructions may include protocols for performing any one or more of the methods described herein.

[0431] In some embodiments and settings, the kit may include materials for producing the final payload-supported polypeptide complex according to the Disclosure. In some embodiments, the kit may include a predetermined amount of “donor” precursor polypeptide complex and / or a predetermined amount of “acceptor” precursor polypeptide complex. In some embodiments, the kit may include reagents for producing the final payload-supported polypeptide complex according to the Disclosure from the “donor” and “acceptor” precursor polypeptide complexes. In some embodiments, the kit may include instructions for producing the final payload-supported polypeptide complex according to the Disclosure from the “donor” and “acceptor” precursor polypeptide complexes, for example, according to the method described herein. [table] TIFF2026509081000011.tif254170TIFF2026509081000012.tif255170TIFF20265090810 00013.tif249170TIFF2026509081000014.tif254170TIFF2026509081000015.tif208170 TIFF2026509081000016.tif245170TIFF2026509081000017.tif220170TIFF20265090810 00018.tif254170TIFF2026509081000019.tif254170TIFF2026509081000020.tif176170 [Brief explanation of the drawing]

[0432] [Figure 1]This is a schematic diagram illustrating the concept of chain exchange for attaching a payload. A set of one-arm antibodies is shown exemplarily. Different binders have various sequences in the Fab region. The upper right (circle) shows a single conjugation reaction with an Fc-only dimer. The conjugated donor molecule is then applied to exchange reactions with each of the different binders (acceptors). After chain exchange, the mixture is applied to an affinity C-tagged column that binds to all dummy dimers, aggregates, or unreacted starting material molecules containing C tags. The defined labeled antibody, which is the product, is present in the flow-through with high product purity. [Figure 2] Figures 2A-2D: Schematic diagrams, graphs, images, and bar graphs regarding chain exchange-mediated attachment of dyes derived from NHS-lysine conjugate donor modules. (2A) Two reactant molecules were mixed at equimolar concentrations in PBS under mild reducing conditions (20x molar access TCEP). The mixture was maintained at 37°C for 3 hours with shaking. The reactants were then loaded into a C-tag affinity chromatograph and the flow-through was collected. (2B) The labeled product had a purity of over 98%, and capillary electrophoresis (CE-SDS) revealed defined bands of expected size under both (non-)reducing conditions. HC = heavy chain, LC = light chain, Ab = antibody. (2C) Exemplarily, two different formats were used: a Fab-Fc molecule containing the CH1 domain and the Ckappa domain, and a format with scFv as the targeted entity (VH+VL only). (2D) Two binders (C6.5, F5) in different formats (Fab-Fc and scFv-Fc) and a positive control 4D5-8 (derived from trastuzumab in Fab-Fc format) were used. All molecules had the same DAR without significant difference in labeling. As expected, the payload-Fc loaded molecules had more than twice the amount of payload attached. [Figure 3]Figures 3A and 3B: Schematic diagrams, graphs, and bar graphs relating to the internal migration capabilities of different Her2 binders. Strong chain exchange-mediated adhesion of dyes derived from NHS-lysine conjugate donor modules. (3A) To validate the concept, five different clones / formats were labeled with pH-dependent dyes (pHab). The products were applied to Her2-positive cell lines. After internal migration, the dyes accumulated and acquired activity in the acidic environment of endosomes, thereby allowing for the measurement of antibody-mediated internal migration. (3B) All binders internalized as reflected by increased PE signals compared to isotype controls. Absolute internal migration (intracellular volume of dye) and relative internal migration (ratio to binding signal) are shown. [Figure 4] Schematic diagram and image of chain exchange-mediated attachment of horseradish peroxidase. Horseradish peroxidase (HRP) was attached to the EGFR-binding antibody C225. Western blotting shows the suitability of this binder for detecting denatured (SDS-PAGE) EGFR and the activity of the attached enzyme. [Figure 5] Figures 5A and 5B show schematic diagrams, images, and bar graphs illustrating the transport of payloads conjugated to donor Fc in a site-specific manner. Strong chain-exchange mediated attachment of dyes derived from NHS-lysine conjugated donor modules is also shown. (3A) Biotin was conjugated to Fc precursor molecules via site-specific Avi-tagged biotinylation. The functionality of the biotinylated antibody obtained after chain exchange was demonstrated by its application in immunoprecipitation of EGFR from whole A431 cell lysates (magnetic streptavidin beads). (3B) Ruthenium was conjugated to Fc precursor molecules possessing a C-terminal K-tag. The ruthenium-containing Q-tag was attached to the K-tag of donor Fc via transglutaminase. The functionality of the ruthenium-conjugated antibody obtained after chain exchange was demonstrated by its application in an electrochemiluminescence assay (Elecsys / Cobas). [Figure 6]Schematic diagrams and graphs of antibody-payload fusion proteins generated by chain exchange-mediated attachment. An EGFR-binding acceptor molecule based on the C225 (cetuximab) sequence was subjected to chain exchange with a donor chain containing a GFP fusion protein at its C-terminus. FACS analysis demonstrated specific binding to EGFR-expressing A431 cells, and competitive experiments with other published antibodies (P1X, P2X, and mab806) revealed EGFR domain III as the target of C225, consistent with published data (Li et al. 2005). [Examples]

[0433] Example 1: Method 1.1 Expression and Purification of Precursor Molecules The expression plasmid encoded a CMV promoter transcription recombinant antibody derivative that was secreted into the cell culture supernatant as described above (Dengl et al.). A transient HEK-293-Expi system (Thermo Fisher) was used according to the manufacturer's instructions. The cultures were maintained at 37°C for 6 days in a humidified 8% CO2 atmosphere. All proteins described herein were secreted into the culture supernatant (IgG VH secretory leader peptide). The supernatant was separated from the cell contents by centrifugation (3,500 g, 45 min) and filtration (0.22 μm). The antibody derivatives were then concentrated using ProtA (HiTrap® Protein A HP, GE, catalog no. 28989336, Boston, MA, USA) affinity chromatography and size exclusion chromatography (HiLoad® 26 / 600 Superdex® 200, GE, catalog no. 28989336, Boston, MA, USA).

[0434] The polypeptides used in the experimental examples of this disclosure are shown in SEQ ID NOs. 72-103. The following polypeptides were used: Acceptor precursor molecule: [table] TIFF2026509081000021.tif57170[Table] TIFF2026509081000022.tif68170[Table] TIFF2026509081000023.tif68170[Table] TIFF2026509081000024.tif57170

[0435] Donor precursor molecules: [table] TIFF2026509081000025.tif47170[Table] TIFF2026509081000026.tif57170[Table] TIFF2026509081000027.tif57170[Table] TIFF2026509081000028.tif57170[Table] TIFF2026509081000029.tif57170[Table] TIFF2026509081000030.tif57170[Table] TIFF2026509081000031.tif57170[Table] TIFF2026509081000032.tif57170

[0436] 1.2 Payload attachment to replaceable precursor molecules Fc-only precursor molecules were coupled to the payload to function as payload donor entities. This was achieved by (i) site-specific fusion of enhanced GFP (UniProt) containing a G4S-linker to the C-terminus, (ii) site-specific conjugation of biotin by Avi tag technology (BirA Bulk Kit, Avidity, manufacturer's instructions), (iii) random amine-reactive labeling with pH-sensitive dyes (Promega, catalog no. G9845, manufacturer's instructions including DAR calculation), and (iv) random amine-reactive labeling with horseradish peroxidase (EZ-Link® Activated Peroxidase Antibody Labeling Kit, Thermo Scientific, catalog no. 31497, manufacturer's instructions). Chemical or enzymatic coupling reactions were performed according to the manufacturer's protocol.

[0437] 1.3 Transfer of payload from donor to acceptor via chain exchange To facilitate polypeptide chain exchange between donor precursor molecules and acceptor precursor molecules, a donor precursor containing a knob-dummy polypeptide is mixed with an acceptor precursor containing the corresponding hole-dummy polypeptide, and conversely, a precursor containing a hole-dummy polypeptide is mixed with an acceptor precursor containing the corresponding knob-dummy polypeptide.

[0438] The donor and acceptor precursor complexes were mixed in equimolar amounts at a concentration of 1 mg / ml in PBS (pH 7.4). A 20-fold molar excess of TCEP was added to reduce the disulfide crosslinks in the hinge region and initiate the exchange reaction. The mixture was held at 37°C and 300 rpm for 3 hours. The resulting exchange product was purified by adsorption of aggregates, unreacted precursors, and dummy dimers via their C-tags on affinity columns (CaptureSelect® C-tagXL Pre-packed Column / Thermo Scientific, catalog no. 494307205). The composition and quality of the product were evaluated by analytical SEC (Superdex 200 analytical size exclusion column, GE Healthcare, Sweden) and capillary electrophoresis (CE-SDS, Caliper Life Sciences) in 200 mM KH2PO4, 250 mM KCl, and pH 7.0 running buffer at 25°C.

[0439] 1.4 Binding assays and internal distribution assays Binding to target cells was evaluated by flow cytometry. Total 3 × 10⁶ 5 Cells were incubated with 200 nM antibody derivatives in FACS buffer (PBS containing 2% FCS) at 37°C for 1 hour. The cells were then washed twice with PBS, and phycoerythrin intensity was analyzed using a FACS Canto II instrument (BD biosciences). FloJo (BD) software was used for data analysis and visualization. For epitope binning assays, 200 nM competitive IgG (P1X, P2X, and mab806) binding to known epitopes on EGFR domains I, II, and III were added to separate wells, and FACS analysis was performed as described above. FITC intensity was measured using a FACS Canto II instrument. To study internal migration, 1 × 10⁶ cells were used. 5Each SK-BR-3 cell (ATCC) was seeded in a flat-bottomed 96-well plate and treated with 200 μl of final volume of 500 nM Her-2-bound clones labeled with pHAb. The mixture was maintained at 37°C for 24 hours under a humidified 8% CO2 atmosphere. The cells were then detached with 100 μL of Accutase (Pan Biotech, catalog no. P10-21100), washed twice with PBS, and analyzed via cytometry as described above. GraphPad Prism 7 software (GraphPad Software, San Diego, CA, USA) was used for bar graph visualization.

[0440] 1.5 Immunoprecipitation and Western blot 3 x 10 6A431 cells were resuspended on ice in 1 ml of RIPA lysis and extraction buffer (Thermo Scientific, catalog no. 89900). Cell debris was removed by centrifugation at 15000 g for 15 minutes, and the supernatant was collected. EGFR molecules were pulled down with biotinylated C225 antibody using Pierce® streptavidin magnetic beads (Thermo Scientific, catalog no. 88816, applied according to the manual). Elution from the beads was performed at 95°C for 5 minutes using 50 μl of NuPAGE® LDS Sample Buffer (Thermo-Fisher, catalog no. NP0007). After removing the magnetic beads, 20 μl was loaded onto a 4-12% Bis-Tris Gel (Invitrogen, catalog no. NP0322BOX) and run at 180 V for 60 minutes. Proteins were blotted using Trans-Blot Turbo Mini 0.2μm PVDF Transfer Packs (Bio-Rad, catalog number 1704156) and the Trans-Blot Turbo Transfer System (Bio-Rad) according to the manufacturer's instructions for high molecular weight species. The membranes were then blocked in blocking buffer (1×TBS, 0.05% Tween20, 5% skim milk) for 30 minutes. The membranes were then incubated overnight in blocking buffer containing the primary anti-EGFR antibody (Abcam, ab264540, 1:1000). The following day, the membranes were washed with TBS-T for 3×5 minutes and incubated in blocking buffer with the secondary antibody (polyclonal goat anti-mouse immunoglobulin / HRP; Agilent Dako, catalog number P044701-2, 1:1500) at RT for 1 hour. After three TBS-T washes, chemiluminescence was detected using SuperSignal® West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, catalog number 34579) and the Gel Doc XR+ Gel Documentation System. Western blotting procedures were similarly performed to test the functionality of C225-HRP.The A431 solution was loaded onto the gel, blotting was performed accordingly, and then C225-HRP was applied in blocking buffer at a concentration of c=0.24 μg / mL, using the readout described above.

[0441] Example 2: Results and Discussion 2.1 Payload redirection chain exchange The principle of the payload redirection chain exchange concept is shown in Figure 1. As previously described by Dengl et al. 2020, different binder modules (black, dark gray, light gray) become interchangeable by possessing a partially destabilized knob-into-hole CH3 interface with repulsive charges. These derivatives, having different binding (Fab / Fv) regions, represent acceptor entities of the payload to which they are attached. Payload donor entities are complementary (partially destabilized) knob-into-hole Fc-only precursors that are conjugated to one or more payloads in a site-specific or random manner.

[0442] When acceptor and donor entities are mixed under slightly reducing conditions for the limited reduction of hinged disulfide (see the materials and methods section of Dengl et al. 2020), an exchange reaction is triggered, driven by repulsive charges at the precursor interface. This reaction attaches the payload to the binder, along with a dummy dimer as a byproduct. The dummy dimer, as well as the donor and acceptor precursors (but not the ADC product), have a C-terminal C-tag and can be removed from the reaction mixture by adsorption to a C-Tag affinity column. The flow-through of these columns contains the binder coupled with the payload. Conjugate-mediated variation between the resulting ADCs is excluded due to the fact that the exchange reaction transports one defined conjugate as the donor to a set of many different acceptor-binders. All products have different binders but identical payloads and drug-to-antibody ratios (DARs) and are attached to the same position on the ADC. Defined payload attachment by exchange can be performed using various payloads attached to the donor module by different means. This includes "random" attachment to donors (which are then converted into defined ADC products), and attachment via site-specific payload conjugation or fusion to donor entities, as described below.

[0443] 2.2 A defined set of ADCs generated from random NHS conjugated payload donor modules Conjugation of NHS-modified payloads to lysine residues exposed on the protein surface is a robust, first-generation, and promising technique for ADC production. Because multiple lysine residues are scattered on the antibody surface, sometimes directly or in close proximity to the antigen-binding site, this type of conjugation can be considered "random." Therefore, it is difficult to produce a set of antibodies with different sequences that conjugate to the NHS-payload in an equivalent manner (DAR & position) where all molecules retain complete binding functionality. Chain exchange techniques overcome the common obstacles of heterogeneous conjugation patterns and potentially impaired binding regions. Figure 2 illustrates how NHS conjugation (with a payload randomly assigned to a donor-Fc) is transformed by exchange-mediated attachment to a set of different antibodies with identical payloads (DAR) and unmodified binding regions. In this two-step method, the payload is first conjugated to an exchange donor rather than directly fused to the antibody of interest. Then, the transport of the exchange module in the second step is constant among all the antibodies (acceptors) to which the payload is attached.

[0444] As an example of this approach, different Her2-targeted acceptor molecules were mixed in equimolar concentrations with Fc-on-leadner molecules pre-labeled with pH-sensitive dyes by random conjugation to reactive amine groups. The reaction buffer was standard PBS containing 20-fold molar concentration of TCEP (reducing agent) to decompose the hinge-disulfide. After orbital shaking at 300 rpm for 3 hours at 37°C, the mixture was applied to a C-tagged resin. Flow-through samples contained a labeled product of high purity (>95%) and expected composition (Figure 2B). To demonstrate that the reaction is not affected by the binder sequence or the format of the target-binding acceptor (Figure 2C), three different Her2 binders and Her2 binders of two different formats were compared. Figure 2D shows that all binders of all formats (Fab-Fc containing CH1 and Ck; scFv-Fc) were labeled to the same degree. All acceptor molecules had the same exchange interface, and consequently, the DAR was the same in all five examples. Figure 2D also reveals that the initial donor payload-Fc precursor possessed approximately twice the number of fluorophores found in the product. This confirms an exchange reaction mechanism in which half of the donor molecule is transported to the acceptor and the other half becomes a dummy dimer byproduct.

[0445] When comparing the functionality and / or applicability of different antibodies, it is crucial to produce sets of ADCs with identical labeling and DARs while ensuring unimpaired binding regions. One parameter known to modulate the effectiveness of ADCs using cytotoxic payloads is the ability to internalize the payload and thereby deliver it to target cells (Maass et al. 2016; Tang et al. 2019). pH-sensitive dyes attached to different Her2 binders (Figure 2) illuminate upon internalization into cells and transport to the endo- / lysosomal pathway where they encounter an acidic environment (Figure 3A). A prerequisite for applying ADCs with this dye to compare and quantify internalization is that the different binders have the dye in equivalent manner without affecting their binding regions. This is difficult to achieve by individual NHS conjugations but is ensured by our simple and robust chain exchange procedure. The applicability of the ADCs produced thereby for comparing internalization of different Her2 binders and formats is shown in Figure 3.

[0446] 2.3 Defined antibody-enzyme conjugates generated from random conjugate donor modules The conversion of random conjugates to rather defined molecules with free binding domains can be achieved not only for small molecular weight payloads such as dyes, but also for attaching larger molecules, i.e., proteins. Figure 4 shows an NHS-lysine conjugate to a donor precursor of the enzyme horseradish peroxidase (HRP). Subsequent exchange reaction causes half of the enzyme-containing donor to attach to an acceptor molecule, in this case an EGFR-binding antibody (clone C225). The combined functionality of both the enzyme and the binding domain was demonstrated by applying the conjugate as a Western blot detection reagent. Figure 4 shows that the resulting anti-EGFR-HRP conjugate detects EGFR in all-blotted cell lysates of A431 cells with high specificity and low background. Therefore, chain exchange-mediated ADC generation is not limited to the attachment of small compounds but is also applicable to large molecular payloads.

[0447] 2.4 ADC generation by payload transport from site-specific conjugate donor modules Chain exchange technology is not limited to the conversion of random NHS conjugates to defined ADCs with intact binding domains. It also enables the effective transport of site-specific donor-attached payloads to different sets of binder acceptors. An example of such applications using donors possessing site-specific modifications is the transglutaminase-mediated attachment of biotinylation and labeling of antibody derivatives. Figure 5 shows donor Fc portions with an Avi tag fused to the N-terminus or C-terminus for site-specific (via birA, see M&M) biotinylation. These can be transported to an acceptor binder and result in a predetermined mono(or bi)biotinylated antibody with an intact antigen-binding domain. These biotinylated antibody derivatives can be used in a variety of assays, such as binder immobilization (e.g., on an SPR chip) and antigen capture, and are defined reagents that can function as general-purpose assay reagents. One application example (antigen pulldown) is shown in Figure 5A.

[0448] Transglutaminase-mediated attachment is another method for enzyme-mediated site-specific conjugation compatible with our exchange technique. Figure 5B shows an example where ruthenium is attached to a transglutaminase site located at the C-terminus of a donor Fc (from a K-tag on ruthenium to a Q-tag on Fc, see M&M). Thus, chain exchange is a simple and robust means of generating many ruthenium-labeled antibody derivatives from a single "stock reagent" that function as highly sensitive detection reagents in electrochemiluminescence (ECL)-based assays (Figure 5B).

[0449] 2.5 Antibody-payload fusion protein generated by chain exchange-mediated adhesion Chain exchange technology is not limited to the attachment of chemically or enzymatically bound payloads. The payload can also be site-specifically positioned relative to the donor Fc as a fusion. The fused Fc is then transported to a binding acceptor to obtain an “ADC-like” antibody-payload combination. This application is particularly well suited for attaching defined fusion partners to many different antibodies in a high-throughput manner. To highlight the versatility of the concept, the inventors produced a payload donor in the form of a C-terminal fusion with enhanced GFP (approximately 30 kDa). This donor molecule behaved well (soluble, low aggregation) and could be expressed in good yields similar to those of a normal antibody. Thus, this protein can be produced as a general-purpose “stock reagent” for exchange-mediated attachment to any exchangeable antibody derivative. The GFP fusion functioned as a payload transport donor in the same manner as random or site-specific donor-payload conjugates. Thus, the fusion protein format did not interfere with the exchange, resulting in effective transport of the fluorescent protein to the acceptor molecule.

[0450] An example of payload transport in the form of a fusion protein is shown in Figure 6. The EGFR-binding acceptor module (C225 / cetuximab-derived sequence) was replaced to generate a GFP fusion derivative, which was then used in a FACS experiment.

[0451] The results of these analyses demonstrate that such molecules are applicable as specific detection reagents in fluorescence readout assays, including FACS analysis (Figure 6). The exchange reaction can generate fluorescent fusion protein derivatives of different antibodies in a simple and robust manner. A common application requiring a comparable set of different antibodies labeled and directed at the same antigen is "epitope binning" (Abdiche et al. 2012). Figure 6 shows that exchange-generated antibody-GFP fusions can be applied to such tasks, demonstrating the conceptual suitability for epitope binning applications.

[0452] References Abdiche,Y.N.,K.C.Lindquist,D.M.Stone,A.Rajpal,and J.Pons.2012.‘Label-free epitope binning assays of monoclonal antibodies enable the identification of antigen heterogeneity’,J Immunol Methods,382:101-16. Akkapeddi,Padma,Saara-Anne Azizi,Allyson M.Freedy,Pedro M.S.D.Cal,Pedro M.P.Gois,and Goncalo J.L.Bernardes.2016.‘Construction of homogeneous antibody-drug conjugates using site-selective protein chemistry’,Chemical science,7:2954-63. Beck,Alain,Liliane Goetsch,Charles Dumontet,and Nathalie Corvaia.2017.‘Strategies and challenges for the next generation of antibody-drug conjugates’,Nature Reviews Drug Discovery,16:315-37. Dengl,S.,K.Mayer,F.Bormann,H.Duerr,E.Hoffmann,B.Nussbaum,M.Tischler,M.Wagner,A.Kuglstatter,L.Leibrock,C.Buldun,G.Georges,and U.Brinkmann.2020.‘Format chain exchange(FORCE)for high-throughput generation of bispecific antibodies in combinatorial binder-format matrices’,Nat Commun,11:4974. Hussain,Ahmad Fawzi,Armin Grimm,Wenjie Sheng,Chaoyu Zhang,Marwah Al-Rawe,Karen Brautigam,Mobarak Abu Mraheil,Felix Zeppernick,and Ivo Meinhold-Heerlein.2021.‘Toward Homogenous Antibody Drug Conjugates Using Enzyme-Based Conjugation Approaches’,Pharmaceuticals,14:343. Li,Shiqing,Karl R.Schmitz,Philip D.Jeffrey,Jed J.W.Wiltzius,Paul Kussie,and Kathryn M.Ferguson.2005.‘Structural basis for inhibition of the epidermal growth factor receptor by cetuximab’,Cancer Cell,7:301-11. Maass,Katie F.,Chethana Kulkarni,Alison M.Betts,and K.Dane Wittrup.2016.‘Determination of Cellular Processing Rates for a Trastuzumab-Maytansinoid Antibody-Drug Conjugate(ADC)Highlights Key Parameters for ADC Design’,The AAPS journal,18:635-46. Mohlmann,S.,P.Bringmann,S.Greven,and A.Harrenga.2011.‘Site-specific modification of ED-B-targeting antibody using intein-fusion technology’,BMC Biotechnol,11:76. Nath,N.,B.Godat,C.Zimprich,S.J.Dwight,C.Corona,M.McDougall,and M.Urh.2016.‘Homogeneous plate based antibody internalisation assay using pH sensor fluorescent dye’,J Immunol Methods,431:11-21. Patterson,James T.,Shigehiro Asano,Xiuling Li,Christoph Rader,and Carlos F.Barbas,3rd.2014.‘Improving the serum stability of site-specific antibody conjugates with sulfone linkers’,Bioconjugate chemistry,25:1402-07. Sadiki,Amissi,Shefali R Vaidya,Mina Abdollahi,Gunjan Bhardwaj,Michael E Dolan,Harpreet Turna,Varnika Arora,Athul Sanjeev,Timothy D Robinson,Andrea Koid,Aashka Amin,and Zhaohui Sunny Zhou.2020.‘Site-specific conjugation of native antibody’,Antibody Therapeutics,3:271-84. Steffen,W.,F.C.Ko,J.Patel,V.Lyamichev,T.J.Albert,J.Benz,M.G.Rudolph,F.Bergmann,T.Streidl,P.Kratzsch,M.Boenitz-Dulat,T.Oelschlaegel,and M.Schraeml.2017.‘Discovery of a microbial transglutaminase enabling highly site-specific labeling of proteins’,J Biol Chem,292:15622-35. Strop,Pavel,Shu-Hui Liu,Magdalena Dorywalska,Kathy Delaria,Russell G Dushin,Thomas-Toan Tran,Wei-Hsien Ho,Santiago Farias,Meritxell Galindo Casas,Yasmina Abdiche,Dahui Zhou,Ramalakshmi Chandrasekaran,Caroline Samain,Carole Loo,Andrea Rossi,Mathias Rickert,Stellanie Krimm,Teresa Wong,Sherman Michael Chin,Jessica Yu,Jeanette Dilley,Javier Chaparro-Riggers,Gary F Filzen,Christopher J O’Donnell,Fang Wang,Jeremy S Myers,Jaume Pons,David L Shelton,and Arvind Rajpal.2013.‘Location Matters:Site of Conjugation Modulates Stability and Pharmacokinetics of Antibody Drug Conjugates’,Chemistry&Biology,20:161-67. Su,Dian,and Donglu Zhang.2021.‘Linker Design Impacts Antibody-Drug Conjugate Pharmacokinetics and Efficacy via Modulating the Stability and Payload Release Efficiency’,Frontiers in Pharmacology,12. Sun,X.,J.F.Ponte,N.C.Yoder,R.Laleau,J.Coccia,L.Lanieri,Q.Qiu,R.Wu,E.Hong,M.Bogalhas,L.Wang,L.Dong,Y.Setiady,E.K.Maloney,O.Ab,X.Zhang,J.Pinkas,T.A.Keating,R.Chari,H.K.Erickson,and J.M.Lambert.2017.‘Effects of Drug-Antibody Ratio on Pharmacokinetics,Biodistribution,Efficacy,and Tolerability of Antibody-Maytansinoid Conjugates’,Bioconjugate chemistry,28:1371-81. Tang,Haichao,Yan Liu,Zhaojin Yu,Mingli Sun,Lu Lin,Wensi Liu,Qiang Han,Minjie Wei,and Ying Jin.2019.‘The Analysis of Key Factors Related to ADCs Structural Design’,Frontiers in Pharmacology,10. Tsuchikama,Kyoji,and Zhiqiang An.2018.‘Antibody-drug conjugates:recent advances in conjugation and linker chemistries’,Protein&Cell,9:33-46.

Claims

1. A method for producing polypeptide complexes, below: (1) A first polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain, The first polypeptide complex is formed by an interaction between the first polypeptide and the second polypeptide, the interaction including an association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide, The CH3 domain of the first polypeptide includes a knob modification, and the CH3 domain of the second polypeptide includes a hole modification. The CH3 domain of the first polypeptide or the CH3 domain of the second polypeptide includes destabilizing modifications to destabilize the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide, The first polypeptide and / or the second polypeptide further comprises a payload portion, (2) A second polypeptide complex comprising a third polypeptide containing a CH3 domain and a fourth polypeptide containing a CH3 domain, The second polypeptide complex is formed by an interaction between the third polypeptide and the fourth polypeptide, the interaction comprising an association between the CH3 domain of the third polypeptide and the CH3 domain of the fourth polypeptide, The CH3 domain of the third polypeptide includes a knob modification, and the CH3 domain of the fourth polypeptide includes a hole modification. The CH3 domain of the third polypeptide or the CH3 domain of the fourth polypeptide is destabilized with a second polypeptide complex, which includes destabilization modifications to destabilize the association between the CH3 domain of the third polypeptide and the CH3 domain of the fourth polypeptide. Incubate, To form a third polypeptide complex comprising the first polypeptide and the fourth polypeptide, and / or a fourth polypeptide complex comprising the second polypeptide and the third polypeptide, and This includes recovering the third polypeptide complex and / or the fourth polypeptide complex, The destabilizing modification of the CH3 domain of the first polypeptide or the second polypeptide does not destabilize the association between the first polypeptide and the fourth polypeptide, nor destabilizes the association between the second polypeptide and the third polypeptide. A method for destabilizing the CH3 domain of the third polypeptide or the fourth polypeptide without destabilizing the association between the first polypeptide and the fourth polypeptide, and without destabilizing the association between the second polypeptide and the third polypeptide.

2. The method according to claim 1, wherein the destabilizing modification of the CH3 domain of the first polypeptide or the second polypeptide stabilizes the association between the CH3 domain of the first polypeptide and the CH3 domain of the fourth polypeptide, and / or stabilizes the association between the CH3 domain of the second polypeptide and the CH3 domain of the third polypeptide.

3. The method according to claim 1 or 2, wherein the destabilizing modification of the CH3 domain of the third polypeptide or the fourth polypeptide stabilizes the association between the CH3 domain of the first polypeptide and the CH3 domain of the fourth polypeptide, and / or stabilizes the association between the CH3 domain of the second polypeptide and the CH3 domain of the third polypeptide.

4. The method according to any one of claims 1 to 3, wherein the first polypeptide includes a payload portion and the fourth polypeptide includes a functional portion, or the second polypeptide includes a payload portion and the third polypeptide includes a functional portion.

5. A method according to any one of claims 1 to 4, (a) The CH3 domain of the first polypeptide contains 370E, the CH3 domain of the fourth polypeptide contains 357K, and optionally, the CH3 domain of the second polypeptide contains 357E, the CH3 domain of the third polypeptide contains 370K, or (b) A method wherein the CH3 domain of the first polypeptide comprises 370K, the CH3 domain of the fourth polypeptide comprises 357E, and optionally, the CH3 domain of the second polypeptide comprises 357K, and the CH3 domain of the third polypeptide comprises 370E.

6. A method according to any one of claims 1 to 5, (a) The CH3 domain of the first polypeptide comprises 366W and 370E, the CH3 domain of the second polypeptide comprises 407V, 366S and 368A, the CH3 domain of the third polypeptide comprises 366W, and the CH3 domain of the fourth polypeptide comprises 407V, 366S, 368A and 357K, or (b) The CH3 domain of the first polypeptide comprises 366W, 370E and 354C, the CH3 domain of the second polypeptide comprises 407V, 366S and 368A, the CH3 domain of the third polypeptide comprises 366W, the CH3 domain of the fourth polypeptide comprises 407V, 366S, 368A, 357K and 349C, or (c) The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 24, the CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 49, the CH3 domain of the third polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 48, the CH3 domain of the fourth polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 25, or (d) A method wherein the CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 26, the CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 49, the CH3 domain of the third polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 48, and the CH3 domain of the fourth polypeptide has at least 70% amino acid sequence identity with SEQ ID NO:

27.

7. The method according to any one of claims 1 to 6, wherein the payload portion is a detectable portion, a fluorescent portion, a luminescent portion, a radiopaque / contrast agent, a radiolabel, an immunodetectable portion, a portion having detectable activity, an enzyme portion, a drug portion, or a cytotoxic portion, or comprises the same.

8. The method according to any one of claims 1 to 7, wherein the functional portion is a binding portion, an antibody or its target-binding fragment or derivative, a target-binding peptide / polypeptide, a target-binding nucleic acid, a detectable portion, a fluorescent portion, a luminescent portion, a radiopaque / contrast agent, a radiolabel, an immunodetectable portion, a portion having detectable activity, an enzyme portion, a drug portion, or a cytotoxic portion, or comprises the same.

9. The method according to any one of claims 1 to 8, wherein the first polypeptide, the second polypeptide, the third polypeptide and / or the fourth polypeptide further comprises a CH2 domain and / or a hinge region.

10. A polypeptide complex comprising the third polypeptide complex or the fourth polypeptide complex, produced by the method described in any one of claims 1 to 9.

11. A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain, The polypeptide complex is formed by an interaction between the first polypeptide and the second polypeptide, the interaction including an association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide, The CH3 domain of the first polypeptide includes a knob modification, and the CH3 domain of the second polypeptide includes a hole modification. The CH3 domain of the first polypeptide includes a destabilizing modification, and the CH3 domain of the second polypeptide includes a destabilizing modification. The destabilizing modification of the CH3 domain of the first polypeptide does not destabilize the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide, and the destabilizing modification of the CH3 domain of the second polypeptide does not destabilize the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide. A polypeptide complex comprising the first polypeptide or the second polypeptide, further comprising a payload portion.

12. The polypeptide complex according to claim 11, wherein the first polypeptide and / or the second polypeptide further comprises a functional moiety.

13. The polypeptide complex according to claim 11 or 12, wherein the destabilizing modification of the CH3 domain of the first polypeptide stabilizes the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide, and / or the destabilizing modification of the CH3 domain of the second polypeptide stabilizes the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide.

14. A polypeptide complex according to any one of claims 11 to 13, wherein the first polypeptide comprises a payload portion and the second polypeptide comprises a functional portion, or the first polypeptide comprises a functional portion and the second polypeptide comprises a payload portion.

15. A polypeptide complex according to any one of claims 11 to 14, (a) The CH3 domain of the first polypeptide comprises 366W and 370E, and the CH3 domain of the second polypeptide comprises 407V, 366S, 368A and 357K, or (b) The CH3 domain of the first polypeptide comprises 366W, 370E and 354C, and the CH3 domain of the second polypeptide comprises 407V, 366S, 368A, 357K and 349C, or (c) The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 24, and the CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 25, or (d) A polypeptide complex wherein the CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 26, and the CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO:

27.

16. The polypeptide complex according to any one of claims 11 to 15, wherein the payload portion is a detectable portion, a fluorescent portion, a luminescent portion, a radiopaque / contrast agent, a radiolabel, an immunodetectable portion, a portion having detectable activity, an enzyme portion, a drug portion, or a cytotoxic portion, or comprises the same.

17. The polypeptide complex according to any one of claims 11 to 16, wherein the functional portion is a binding portion, an antibody or its target-binding fragment or derivative, a target-binding peptide / polypeptide, a target-binding nucleic acid, a detectable portion, a fluorescent portion, a luminescent portion, a radiopaque / contrast agent, a radiolabel, an immunodetectable portion, a portion having detectable activity, an enzyme portion, a drug portion, or a cytotoxic portion, or comprises the same.

18. The polypeptide complex according to any one of claims 1 to 17, wherein the first polypeptide and / or the second polypeptide further comprises a CH2 domain and / or a hinge region.

19. A polypeptide complex comprising a first polypeptide containing a CH3 domain and a second polypeptide containing a CH3 domain, The polypeptide complex is formed by an interaction between the first polypeptide and the second polypeptide, the interaction including an association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide. The CH3 domain of the first polypeptide includes a knob modification, and the CH3 domain of the second polypeptide includes a hole modification. The CH3 domain of the first polypeptide and / or the CH3 domain of the second polypeptide include destabilizing modifications to destabilize the association between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide, A polypeptide complex comprising the first polypeptide and / or the second polypeptide, further comprising a payload portion.

20. A polypeptide complex according to claim 19, (a) The CH3 domain of the first polypeptide contains 370E, and the CH3 domain of the second polypeptide contains 357E, or (b) A polypeptide complex in which the CH3 domain of the first polypeptide contains 370K and the CH3 domain of the second polypeptide contains 357K.

21. A polypeptide complex according to either claim 19 or claim 20, (a) The CH3 domain of the first polypeptide comprises 366W and 370E, and the CH3 domain of the second polypeptide comprises 407V, 366S and 368A, or (b) The CH3 domain of the first polypeptide comprises 366W, and the CH3 domain of the second polypeptide comprises 407V, 366S, 368A and 357K, or (c) The CH3 domain of the first polypeptide comprises 366W, 370E and 354C, and the CH3 domain of the second polypeptide comprises 407V, 366S and 368A, or (d) The CH3 domain of the first polypeptide comprises 366W, and the CH3 domain of the second polypeptide comprises 407V, 366S, 368A, 357K and 349C, or (e) The CH3 domain of the first polypeptide comprises 366W and 370E, and the CH3 domain of the second polypeptide comprises 407V, 366S, 368A and 349C, or (f) The CH3 domain of the first polypeptide comprises 366W and 354C, and the CH3 domain of the second polypeptide comprises 407V, 366S, 368A and 357K, or (g) The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 48, and the CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 25, or (h) The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 26, and the CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 49, or (i) The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 48, and the CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 27, or (j) The CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 24, and the CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 51, or (k) A polypeptide complex wherein the CH3 domain of the first polypeptide has at least 70% amino acid sequence identity with SEQ ID NO: 50, and the CH3 domain of the second polypeptide has at least 70% amino acid sequence identity with SEQ ID NO:

25.

22. The polypeptide complex according to any one of claims 19 to 21, wherein the payload portion is a detectable portion, a fluorescent portion, a luminescent portion, a radiopaque / contrast agent, a radiolabel, an immunodetectable portion, a portion having detectable activity, an enzyme portion, a drug portion, or a cytotoxic portion, or comprises the same.

23. The polypeptide complex according to any one of claims 19 to 22, wherein the first polypeptide and / or the second polypeptide further comprises a CH2 domain and / or a hinge region.

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