Methods of producing dual specific proteins

Abstract

The present application relates to methods of producing dual specific proteins. Provided herein is a method of producing a heterodimeric protein comprising two different IgG CH3 domains capable of forming a CH3-CH3 interface. In addition, provided herein are isolated heterodimeric proteins obtained by the method. Also provided herein are isolated heterodimeric antibodies comprising a first IgG CH3 domain and a second IgG CH3 domain wherein the first CH3 domain and the second CH3 domain are capable of forming a CH3-CH3 interface, as well as pharmaceutical compositions comprising the isolated heterodimeric proteins.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202380092161.5 entitled "Method for generating dual-specific proteins", filed on December 22, 2023. Technical Field

[0002] This document provides a method for generating a heterodimeric protein (heterodimeric protein, heterodimer protein) comprising two distinct IgG CH3 domains capable of forming a CH3-CH3 interface. Furthermore, this document provides the isolated heterodimeric protein obtained by said method. This document also provides an isolated heterodimeric antibody (heterodimeric antibody) comprising a first IgG CH3 domain and a second IgG CH3 domain, wherein the first CH3 domain and the second CH3 domain are capable of forming a CH3-CH3 interface capable of generating multiple specific binding domains; and a pharmaceutical composition comprising the isolated heterodimeric protein having multiple binding specificities. Background Technology

[0003] Monospecific antibodies play a crucial role as therapeutic molecules for a variety of diseases, particularly cancer. Monospecific antibodies bind to a single, specific region or epitope of an antigen, and for use in therapy, they are typically selected for the desired functional properties (e.g., killing tumor cells, blocking receptor-ligand interactions, or viral neutralization). Monospecific antibodies possess several advantageous properties, such as their ability to be mass-produced and their biophysical and biochemical properties allowing for very detailed analysis to ensure batch-to-batch consistency, which contributes to regulatory acceptability.

[0004] Despite these advantageous properties, monospecific antibodies have several drawbacks associated with their specificity. Therefore, in recent years, bispecific and multispecific antibodies have begun to play an even more significant role, as they have the potential to overcome some of the limitations of monospecific antibody therapy. For example, by combining with target molecules that are present only on tumor cells, they can be used as mediators to target cells with drugs or toxic compounds, as mediators to retarget effector mechanisms to disease-related sites, or as mediators to increase specificity for tumor cells.

[0005] Despite the promising prospects, the production and testing of multispecific antibodies remain challenging. For example, bispecific antibodies based on IgG, consisting of two heavy chains and two light chains, have been produced using various methods. These include fusing cell lines containing two secreting antibodies to create new cell lines or expressing two antibodies in a single cell using recombinant DNA technology. These methods yield a variety of antibody types because the corresponding heavy chains from each antibody can form monospecific dimers (also known as homodimers or homodimeric antibodies) containing two identical paired heavy chains with the same specificity, and bispecific dimers (also known as heterodimers or heterodimeric antibodies) containing two different paired heavy chains with different specificities. Furthermore, the light and heavy chains from each antibody can randomly pair up to form inappropriate, nonfunctional combinations. This problem is known as heavy and light chain mismatch. This can be addressed by selecting antibodies that share a common light chain for expression as bispecific antibodies. When using a common light chain, expressing two heavy chains and one common light chain in a single cell can result in the production of three different antibody species (i.e., two monospecific “parental” antibodies and a bispecific antibody), thus requiring purification of the bispecific antibody of interest from the resulting antibody mixture.

[0006] While a single cell can generate a substantially single antibody species using heterodimerization techniques that pair bispecific constant regions, obtaining this product requires techniques that restrict the ability to combine different binding domains (e.g., a common light chain binding domain with a non-common light chain antibody). Therefore, there remains a need for multispecific forms that allow combinations of different binding domains (common and non-common light chains) to produce a substantially single product that can be preclinically tested and reliably manufactured for clinical and commercial development. Summary of the Invention

[0007] This disclosure is based on a novel method developed by the inventors for producing heterodimeric proteins, specifically heterodimeric antibodies, which uses a novel Fab-arm exchange method.

[0008] The method described herein is based on a novel Fab-arm exchange method developed by the inventors. This method involves introducing variations (mutations) into the CH3 domain that allow Fab-arm exchanges to occur in all IgG isotypes without destabilizing the core hinge region. As discussed in more detail in the Examples section below, the inventors have demonstrated that Fab-arm exchanges can occur in IgG1 molecules when one CH3 domain contains amino acids 351D and 368E and the other CH3 domain contains amino acids 366K and 351K. Although the examples use these specific amino acids at positions 351, 366, and 368, the method is equally effective when different positively charged amino acid residues at positions 351 and 366 are present in one CH3 domain, and corresponding negatively charged amino acid residues at positions 351 and 368 are present in the other CH3 domain.

[0009] One advantage of this disclosure is that by introducing positively charged amino acids at positions 351 and 366 in its CH3 domain and exchanging its Fab domain with a second antibody containing negatively charged amino acids at positions 351 and 368 in its CH3 domain, the Fab domain can be exchanged with any previously disclosed antibody whose amino acid sequence is given. Currently, over 800 antibodies are known from the World Health Organization's list of International Non-Proprietary Names (INNs), which are either approved or under development, and they can all be combined with another antibody, provided that both antibodies contain two distinct IgG CH3 domains capable of forming a CH3-CH3 interface, wherein one antibody has a CH3 domain with positively charged amino acids at positions 351 and 366, while the other antibody has a CH3 domain with negatively charged amino acids at positions 351 and 368.

[0010] By generating multispecific antibodies containing binding domains of non-shared light chains, expressing such products from single cells results in light chain mismatches, leading to a variety of different antibody species with lost or reduced affinity or specificity. This requires laborious, time-consuming, and inefficient isolation methods to select the desired species. This disclosure now allows for the combination of antibodies with shared light chain binding domains with antibodies with non-shared light chain binding domains, thereby producing multispecific antibody species with relative purity, yield, and efficiency. Furthermore, this disclosure now allows for the combination of antibodies with non-shared light chain binding domains with other antibodies that bind to non-shared light chains. The light chain can be any member of the κ and λ families. The Fab domain can be from any source, including from a shared light chain library.

[0011] In addition to allowing Fab-arm exchange for all IgG forms, this disclosure offers several unexpected advantages. For example, it has been found that, once exposed to reducing and reoxidizing conditions, haptens containing the amino acid combinations 366K and 351K or 351D and 368E primarily remain haptens rather than binding to other haptens with the same residues. As will be apparent to those skilled in the art, this is advantageous in the production of bispecific antibodies, as the tendency of haptens to form homodimers can hinder the efficient production and purification of bispecific antibodies. Furthermore, the presence of homodimers impedes the screening of functional activity for a large number of multispecific antibodies. As shown in more detail in the Examples section of this application, the novel method described herein is likely to be far superior in reducing the generation of potentially undesirable homodimers compared to Fab-arm exchange methods known in the art that rely on the use of the 405L / 409R variant. Specifically, as described in Example 7 of this disclosure, the method of the present invention results in the production of only about 1% homodimers, compared to 5-13% obtained using the 405L / 409R method. Similarly, as shown in Example 7, the method of the present invention can produce a larger amount of heterodimers.

[0012] Therefore, in a first aspect, this document provides a method for generating a heterodimeric protein comprising two different IgG CH3 domains capable of forming a CH3-CH3 interface, the method comprising the following steps: - supply: (a) A first protein comprising a first CH3 domain, wherein the first CH3 domain contains positively charged amino acid residues at positions 351 and 366, and (b) A second protein containing a second CH3 domain, wherein the second CH3 domain contains negatively charged amino acid residues at positions 351 and 368. The numbering is based on the EU numbering. - Incubate proteins (a) and (b) together under reducing conditions to provide a reduced first protein and a reduced second protein; and - The reduced protein is then re-oxidized to obtain a heterodimeric protein.

[0013] In some respects, the first protein and / or the second protein include or are selected from: monomeric proteins, homodimeric proteins and heterodimeric proteins.

[0014] In some respects, the IgG CH3 domain is the IgG1, IgG2, IgG3, or IgG4 CH3 domain.

[0015] In some respects, the IgG CH3 domain is the IgG1 CH3 domain.

[0016] In some respects, the IgG CH3 domain of the first protein and the IgG CH3 domain of the second protein are IgG1.

[0017] In some respects, the IgG CH3 domain of the first protein and the IgG CH3 domain of the second protein are IgG2.

[0018] In some respects, the IgG CH3 domain of the first protein and the IgG CH3 domain of the second protein are IgG3.

[0019] In some respects, the IgG CH3 domain of the first protein and the IgG CH3 domain of the second protein are IgG4.

[0020] In some respects, the IgG CH3 domain is human IgG CH3.

[0021] In some respects, the first and second proteins contain the same hinge region. In some respects, the first and second proteins contain an IgG1 hinge region. In some respects, the first and second proteins contain an IgG2 hinge region. In some respects, the first and second proteins contain an IgG3 hinge region. In some respects, the first and second proteins contain an IgG4 hinge region.

[0022] In some respects, the first and / or second proteins comprise or are selected from: antibodies and haptens, or fragments thereof.

[0023] In some respects, antibodies, haptens, or fragments thereof are human antibodies, haptens, or fragments thereof.

[0024] In some respects, the fragment is a monomeric Fc region or a dimeric Fc region.

[0025] In some respects, the antibody, hapten, or fragment thereof is an IgG1, IgG2, IgG3, or IgG4 antibody, hapten, or fragment thereof. In some respects, the first protein is an antibody containing a first binding specificity, and the second protein may be an antibody containing a second, different binding specificity.

[0026] In some respects, the first protein is an antibody containing binding specificity, and the second protein may be an antibody containing another antibody with different binding specificity.

[0027] In some respects, the first protein is a multispecific antibody (e.g., a bispecific or triple-specific antibody). In some respects, the second protein is a multispecific antibody (e.g., a bispecific or triple-specific antibody). In some respects, both the first and second proteins are multispecific antibodies (e.g., bispecific or triple-specific antibodies).

[0028] In some respects, the first and / or second proteins are homodimeric antibodies.

[0029] In some respects, the first and / or second proteins are heterodimeric antibodies containing a common light chain.

[0030] In some respects, the obtained heterodimeric proteins are heterodimeric antibodies.

[0031] In some respects, the heterodimeric antibody is multivalent, and optionally the multivalent antibody comprises divalent or higher valence, including divalent, trivalent or tetravalent antibodies.

[0032] This disclosure is suitable for generating multivalent polymers known in the art, including but not limited to those using a common light chain. WO2019 / 190327, particularly therein Figure 1 a-1u is incorporated as a reference.

[0033] In some respects, a heterodimeric antibody is a multispecific antibody, optionally wherein the multispecific antibody comprises a divalent or higher valence, and is a bispecific, trispecific, tetraspecific antibody, or has up to a hexavalent valence.

[0034] In some respects, heterodimeric antibodies can contain two distinct light chains.

[0035] In some respects, the first CH3 domain may contain: 351K and 366R, 351R and 366K, 351K and 366K, or 351R and 366R.

[0036] In some respects, the second CH3 domain may contain: 351D and 368E, 351E and 368D, 351D and 368D, or 351E and 368E.

[0037] In some respects, the second CH3 domain may include 351D and 368E, and the first CH3 domain may include 366K and 351K.

[0038] In some respects, the second protein is obtained independently of the first protein.

[0039] In some respects, the conditions for restoration may include: (a) Incubate the protein in the presence of any suitable reducing agent known in the art, optionally wherein the reducing agent comprises or is selected from: 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, β-mercaptoethanol, thioglycolate, cysteamine, homocysteine, penicillamine, and sodium borohydride; and / or (b) Incubate the protein at a pH between 6.0 and 12.0, optionally wherein the pH is between 7.0 and 11.0; and / or (c) The protein is incubated at a redox potential between -150 and -600 mV, optionally wherein the redox potential is between -250 and -400 mV.

[0040] In some respects, the method may also include the steps of enriching and / or separating the heterodimeric protein obtained after re-oxidation.

[0041] In some respects, methods including or selected from: precipitation, centrifugation, filtration, size exclusion chromatography, affinity chromatography, cation and / or anion exchange chromatography and hydrophobic interaction chromatography are used to enrich and / or separate heterodimeric proteins obtained after re-oxidation.

[0042] The method described herein provides a method for generating a heterodimeric antibody comprising two different IgG CH3 domains capable of forming a CH3-CH3 interface, the method comprising the following steps: - supply: (a) A first IgG antibody or half-body, wherein each CH3 domain contains positively charged amino acid residues at positions 351 and 366, and (b) A second IgG antibody or half thereof, wherein each CH3 domain contains negatively charged amino acid residues at positions 351 and 368. The numbering is based on the EU numbering. - Incubate the antibodies and / or halves of (a) and (b) together under conditions sufficient to cause disulfide isomerization of cysteine ​​residues in the core hinge region of the antibody or halves to obtain heterodimeric proteins.

[0043] In another respect, this document provides isolated heterodimeric proteins that are obtainable by the methods of this disclosure.

[0044] In some respects, the heterodimeric protein obtainable by the methods of this disclosure is an IgG antibody.

[0045] In some respects, the heterodimeric proteins obtainable by the methods of this disclosure are multispecific IgG antibodies.

[0046] In some respects, IgG antibodies comprise two light chains with different sequences. In other respects, IgG antibodies include binding domains containing different light chain sequences.

[0047] The isolated heterodimeric antibody comprises a first IgG CH3 domain and a second IgG CH3 domain, wherein the first CH3 domain and the second CH3 domain are capable of forming a CH3-CH3 interface, wherein the first CH3 domain comprises one or more of amino acid variants 366K, 366R, 351K, or 351R, and wherein the second CH3 domain comprises one or more of amino acid variants 351E, 351D, 368E, or 368D, and the heterodimeric antibody further comprises two light chains with different sequences.

[0048] In some respects, the second CH3 domain contains 351D and 368E, and the first CH3 domain contains 366K and 351K.

[0049] In another aspect, this document provides pharmaceutical compositions comprising the isolated heterodimeric protein of this disclosure and a pharmaceutically acceptable carrier.

[0050] In some respects, heterodimeric proteins have been obtained by the methods of this disclosure.

[0051] Throughout the description and claims of this specification, the words “comprising” and “including” and their variations mean “including but not limited to”, and they are not intended to exclude other parts, additions, components, wholes (integers) or steps.

[0052] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. Specifically, when the indefinite article is used, the specification should be understood to include both the plural and the singular unless the context otherwise requires.

[0053] The features, wholes, properties, compounds, chemical parts or groups described in connection with a specific aspect or example of this disclosure should be understood to apply to any other aspect or example described herein, unless incompatible with it.

[0054] Several aspects of this disclosure are described in more detail below. Attached Figure Description

[0055] Aspects of this disclosure are further described below with reference to the accompanying drawings, in which: Figure 1 Top: SDS / PAGE labChip results for reactions #1-#12 in Table 2 without FAE. Bottom: Reactions #1-#12 in Table 2 after FAE.

[0056] Figures 2a-2f HP-CIEX results for reactions #1-#12 in Table 2 without FAE and reactions #1-#12 in Table 2 after FAE. Figures 2a-2f The two graphs above show the results of the reaction without FAE, and the two graphs below show the results of the reaction using FAE. Figure 2a The two images on the left show the results for #1, and the two images on the right show the results for #2. Figure 2b The left and right images show the results for #3 and #4, respectively. Figure 2c The left and right images show the results for #5 and #6, respectively. Figure 2d The left and right images show the results for #7 and #8, respectively. Figure 2e The left and right images show the results for #9 and #10, respectively. Figure 2f The left and right images show the results for #11 and #12, respectively.

[0057] Figures 3a-3d HP-SEC results for reactions #1-#12 in Table 2 without FAE and reactions #1-#12 in Table 2 after FAE. Figures 3a-3d The top three figures show the results of the reaction without FAE, and the bottom three figures show the results of the reaction using FAE. Figure 3a From left to right: Results of #1, #5, and #6. Figure 3b From left to right: the results of #3, #9, and #10. Figure 3c From left to right: the results of #2, #7, and #8. Figure 3d From left to right: Results of #4, #11, and #12.

[0058] Figures 4A-4H Table 3 shows the gel filtration purification and LabChip analysis results for reactions #1-8 after FAE.

[0059] Figure 5 LabChip analysis of FAE products purified by gel filtration in reactions #1-8 in Table 3 under non-reducing conditions (top figure) and reducing conditions (bottom figure).

[0060] Figures 6a-6d CIEX results for samples obtained before FAE, and for samples obtained after FAE and gel filtration. Figure 6a The results of reactions #1 and #2 in Table 3 show the formation of IgG heterodimers before FAE (top figure) and after FAE (bottom figure). Figure 6b The results of reactions #3 and #4 in Table 3 show the formation of IgG heterodimers before FAE (top figure) and after FAE (bottom figure). Figure 6c The results of reactions #5 and #6 in Table 3 show the formation of IgG heterodimers before FAE (top image) and after FAE (bottom image). Figure 6dThe results of reactions #7 and #8 in Table 3 show the formation of IgG heterodimers before FAE (top figure) and after FAE (bottom figure). Detailed Implementation

[0061] Human immunoglobulin G (IgG) antibodies exist in four subclasses with different structural and functional properties. IgG consists of two heavy chain-light chain pairs (half-molecules) linked by inter-heavy chain disulfide bonds located in the hinge region.

[0062] This article provides a method for generating a heterodimeric protein comprising two distinct IgG CH3 domains capable of forming a CH3-CH3 interface. In some respects, the method is an in vitro method.

[0063] As used herein, the term "heterodimeric protein" refers to a protein comprising two monomers, which are different polypeptides covalently or non-covalently linked. One or each of the monomers may pair with a light chain. When used as the first and second proteins provided by methods (a) and (b) of this disclosure, the heterodimeric protein includes a CH3 domain containing positively charged amino acid residues at positions 351 and 366 or negatively charged amino acid residues at positions 351 and 368. The heterodimeric protein obtained by the methods of this disclosure contains positively charged amino acid residues at positions 351 and 366 and negatively charged amino acid residues at positions 351 and 368.

[0064] heterodimeric protein products

[0065] As will be apparent to those skilled in the art, the properties of the heterodimeric protein produced by the methods described in this disclosure will be determined by the starting materials, which are the first and second proteins provided by methods (a) and (b) of this disclosure. By way of example only, if the first and second proteins are fragments of IgG antibodies (e.g., if they contain or consist of IgG CH3 domains), the heterodimeric protein obtained by this method will be a heterodimeric fragment of an IgG antibody (e.g., where the heterodimeric fragment will contain or consist of two IgG CH3 domains). Similarly, if the first and second proteins are IgG antibodies or IgG halves, the resulting heterodimeric protein will be an IgG antibody. In some respects, the first and / or second protein contains a CH3 domain, a hinge region, and a Fab arm. In principle, this technique allows the production of heterodimers, wherein the form of the first and / or second protein may contain any polymerized domains, including but not limited to variable heavy chain domains, CH1 domains, CH2 domains, variable light chains, etc. In some respects, the first protein may contain an antibody-binding domain, and the second protein may contain cytokines, ligands, scFvs, or other domains that offer therapeutic potential (e.g., bifunctional or multifunctional fusion proteins).

[0066] Examples of the first and second proteins are described below.

[0067] In the context of heterodimeric proteins produced by the methods described in this disclosure, the term "heterodimeric protein" refers to a protein comprising two monomers of different polypeptides covalently or non-covalently linked, wherein the two monomers comprise two... differentThe IgG CH3 domains are either the IgG CH3 domains or are composed of them. The IgG CH3 domains differ due to their distinct polypeptide sequences. Specifically, the two distinct IgG CH3 domains differ at least at positions 351, 366, and / or 368. In some respects, one of the two CH3 domains contains positively charged amino acid residues at positions 351 and 366, and the second of the two CH3 domains contains negatively charged amino acid residues at positions 351 and 368. The amino acid residue numbers are based on EU numbers (available from https: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html, last updated on January 20, 2020 at 21:00:03 CET). The CH3 domain containing positively charged amino acid residues at positions 351 and 366 is referred to herein as the “351 / 366 positively charged CH3 domain” or the “first CH3 domain”. Similarly, the CH3 domain containing negatively charged amino acid residues at positions 351 and 368 is referred to in this paper as the "351 / 368 negatively charged CH3 domain" or the "second CH3 domain".

[0068] In this disclosure, when references are made to the 351 / 366 positively charged CH3 domain or the 351 / 368 negatively charged CH3 domain, this specifically refers to the charge of the side chains of residues 351, 366, and 368, and not necessarily to the total charge of the entire CH3 domain. It will be understood that heterodimeric proteins produced by the methods described in this disclosure are heterodimers, at least because they possess two distinct CH3 domains.

[0069] As used herein, the term "CH3 domain" refers to the CH3 domain of an immunoglobulin, specifically the CH3 domain of IgG immunoglobulin. The CH3 domain and its sequence are well known in the art. "IgG immunoglobulin" (also referred to herein as "IgG," "IgG molecule," or "IgG antibody") refers to a polypeptide belonging to the class of antibodies encoded by the immunoglobulin γ gene, which is generally recognized in the art. In humans, the classes of IgG immunoglobulins include subclasses IgG1, IgG2, IgG3, and IgG4. Typically, conventional IgG immunoglobulin is a heterotetramer consisting of two heavy chains and two light chains held together by disulfide bonds (-SS-) in the hinge region. However, in the art, IgG immunoglobulin is often referred to as a dimer (e.g., a homodimer or heterodimer). A dimer is formed from two monomers, each containing a heavy chain and a light chain. The heavy and light chains are held together by disulfide bonds (-SS-). Such monomers are referred to as "half-antibodies" or "semi-antibodies."

[0070] Because different (i.e., 351 / 366 positively charged and 351 / 368 negatively charged) IgG CH3 domains can form CH3-CH3 interfaces, the method described in this disclosure produces heterodimeric proteins. In some respects, the two different IgG CH3 domains preferentially bind to each other, i.e., they tend to bind to each other more readily than to another IgG CH3 domain with the same charge. In other words, the 351 / 368 negatively charged IgG CH3 domain described herein may bind more readily to the 351 / 366 positively charged IgG domain described herein than to the other 351 / 368 negatively charged IgG CH3 domain. Similarly, the 351 / 366 positively charged IgG CH3 domain described herein may bind more readily to the 351 / 368 negatively charged IgG CH3 domain than to the other 351 / 366 positively charged IgG CH3 domain.

[0071] As used herein, the term "CH3-CH3 interface" refers to the binding between two distinct CH3 domains, resulting from the interaction of amino acid residues, specifically at least one interaction between amino acids in the first CH3 domain and amino acids in the second CH3 domain. This interaction occurs, for example, through van der Waals forces, hydrogen bonds, water-mediated hydrogen bonds, salt bridges or other electrostatic forces, attractive interactions between aromatic side chains, disulfide bond formation, or other forces known to those skilled in the art. It will be understood that when two distinct CH3 domains form a CH3-CH3 interface, they form a heterodimeric protein (due to the different sequences of at least the CH3 domains).

[0072] It is known that the interaction between two CH3 domains (such as the two CH3 domains of two separate heavy chains) plays an important role in driving heavy chain dimerization. Therefore, CH3 domains direct the binding of antibody heavy chains, and it is known that the interface between CH3 domains contains more than 20 contact residues from each chain that play a role in the CH3-CH3 interaction (Deisenhofer J., Biochemistry 1981(20)2361-2370; Miller S., J. Mol. Biol. 1990(216)965-973; Padlan, Advances in Protein Chemistry 1996(49) 57-133). Therefore, the CH3 variants of this disclosure (with positively charged amino acid residues at positions 351 and 366 and negatively charged amino acid residues at positions 351 and 368, specific examples of which are discussed in more detail elsewhere in this disclosure) can be used in combination with other antibody domains to produce bispecific or monospecific full-length antibodies. The specificity of antibodies defined by the VH / VL combination generally does not affect heavy chain dimerization behavior driven by the CH3 domain.

[0073] In some respects, the IgG CH3 domain of the heterodimeric protein produced by the methods described in this disclosure does not contain arginine at position 409 and / or does not contain leucine at position 405. In some instances, the IgG CH3 domain of the heterodimeric protein produced by the methods described in this disclosure contains lysine at position 409 and / or contains phenylalanine at position 405 (EU number).

[0074] The heterodimeric protein produced by the methods described herein is a heterodimeric IgG antibody or a heterodimeric fragment of an IgG antibody. In this context, a heterodimeric fragment of an IgG antibody refers to a molecule containing at least two different IgG CH3 domains. In some aspects, the fragment may also contain one or more domains (such as CH2, CH1, VH, CL, and / or VL) and / or a specific binding moiety that are normally present with the IgG antibody.

[0075] In some aspects, a heterodimeric IgG antibody or a heterodimeric fragment of an IgG antibody may contain two IgG CH3 domains, said domains comprising or selected from IgG1, IgG2, IgG3, and IgG4 CH3 domains. In some aspects, each IgG CH3 domain within the heterodimeric IgG antibody or a heterodimeric fragment thereof is an IgG1 CH3 domain. It will be understood that each monomer forming a heterodimeric IgG antibody or a heterodimeric fragment of an IgG antibody does not need to contain an IgG CH3 domain belonging to the same subclass. In some aspects, each CH3 domain of the heterodimeric IgG antibody or a heterodimeric fragment of an IgG antibody belongs to different subclasses. By way of example only, one monomer may contain an IgG1 CH3 domain, while the other may contain an IgG2, IgG3, or IgG4 CH3 domain.

[0076] In some respects, the IgG CH3 domain in the heterodimeric protein produced by the methods described herein is a human IgG CH3 domain (e.g., a human IgG1, IgG2, IgG3, or IgG4 CH3 domain). In some respects, the IgG CH3 domain in the heterodimeric IgG antibody or a heterodimeric fragment of an IgG antibody produced by the methods described herein is a human IgG CH3 domain (e.g., a human IgG1, IgG2, IgG3, or IgG4 CH3 domain).

[0077] In one specific instance, the heterodimeric protein produced by the method described herein is a heterodimeric IgG antibody. In this specific instance, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some respects, it is human IgG1. In some respects, it is human IgG2. In some respects, it is human IgG3. In some respects, it is human IgG4 antibody.

[0078] In another specific example, the heterodimeric protein produced by the method described herein is a heterodimeric fragment of an IgG antibody. In this specific example, the heterodimeric fragment is a heterodimeric fragment of an IgG1, IgG2, IgG3, or IgG4 antibody. In some respects, it is a heterodimeric fragment of human IgG1, IgG2, IgG3, and IgG4 antibodies.

[0079] In some respects, when the heterodimeric protein produced by the methods described herein is a heterodimeric fragment of an IgG antibody, the fragment may contain two IgG CH3 domains and two IgG CH2 domains (e.g., each monomer within the heterodimeric protein may contain both IgG CH3 and IgG CH2 domains).

[0080] In a specific example, the heterodimeric protein produced by the method described herein may contain or consist of an IgG Fc region. In this specific embodiment, the IgG Fc region is the IgG Fc region of an IgG1, IgG2, IgG3, or IgG4 antibody. In some respects, it is the IgG Fc region of human IgG1, IgG2, IgG3, and IgG4 antibodies.

[0081] As used herein, the term "IgG Fc region" refers to the crystallizable C-terminal region of a fragment of the immunoglobulin heavy chain. The human IgG heavy chain Fc region is generally defined as containing amino acid residues from p230 to the C-terminus of the IgG antibody. The residues in the Fc region are numbered according to the EU index. In some respects, the Fc region may include a hinge region. The hinge region (e.g., for IgG1, residues 216-230 according to EU numbers) may extend from the N-terminus of the Fc region. Two monomeric IgG Fc domains are held together by disulfide bonds (-SS-) in the hinge region, thereby contributing to the formation and / or maintenance of heterodimeric proteins. The number of hinge disulfide bonds varies among immunoglobulin subclasses (Papadea and Check 1989). In vivo, the Fc region dimer or heterodimer interacts with the complement system and specific receptors on various cell surfaces. In some respects, the IgG Fc region may contain the IgG1 or IgG2 core hinge region CPPC. Alternatively, the IgG Fc region may contain the IgG3 core hinge region CPRC. Alternatively, the IgG Fc region may include the IgG4 core hinge region (CPSC). As used herein, the term "core hinge region" refers to the four amino acids corresponding to positions 226-229 (EU number) of the human IgG1 antibody.

[0082] In some respects, the first and second proteins contain the same hinge region. In some respects, the first and second proteins contain an IgG1 hinge region. In some respects, the first and second proteins contain an IgG2 hinge region. In some respects, the first and second proteins contain an IgG3 hinge region. In some respects, the first and second proteins contain an IgG4 hinge region.

[0083] In some respects, the heterodimeric proteins produced by the methods described herein are heterodimeric antibodies. Antibodies produced by the methods described herein can have sequences from any source, including mouse and human sequences. Antibodies can consist of sequences from only one source, such as fully human antibodies, or they can have sequences from more than one source, resulting in, for example, chimeric or humanized antibodies. Antibodies intended for therapeutic use are expected to be as close as possible to the natural antibodies of the subject to be treated (e.g., human antibodies for human subjects).

[0084] Antibody binding can be represented by specificity and affinity. Specificity determines which antigen or its epitope is bound by the binding domain. Affinity is a measure of the strength of binding to a specific antigen or epitope.

[0085] As used herein, the term "antigen" refers to a substance or molecule that, when introduced into the body, triggers the immune system to produce antibodies. Antigens can originate from pathogens, tumor cells or other abnormal cells, haptens, or even the body's own structures. At the molecular level, an antigen is characterized by its ability to bind to the antigen-binding site of an antibody. A mixture of antigens can also be considered an "antigen," meaning that those skilled in the art will understand that sometimes lysates of tumor cells or viral particles are represented as "antigens," even though such preparations contain multiple antigenic determinants. An antigen contains at least one, but usually more, epitopes. As used herein, the term "epitaxe" refers to a portion of an antigen that is recognized by the immune system, specifically antibodies, B cells, or T cells. Although epitopes are generally considered to originate from non-self proteins, recognizable sequences derived from the host are also classified as epitopes.

[0086] It will be understood that the heterodimeric proteins (e.g., heterodimeric IgG antibodies or heterodimeric fragments thereof) produced by the methods described herein will consist of two monomers, each containing or consisting of a different IgG CH3 domain (i.e., a 351 / 366 positively charged IgG CH3 domain and a 351 / 368 negatively charged IgG CH3 domain). Furthermore, other differences (i.e., sequence variations) may exist in one or both monomers (e.g., their halves or fragments).

[0087] In some respects, these distinctions can result in multivalent and / or multispecific heterodimeric proteins (such as IgG antibodies or heterodimeric fragments thereof). The term "multivalent" (e.g., a multivalent antibody or a heterodimeric fragment thereof) refers to a single molecule having more than one valence, where "valence" is described as the number of antigen-binding moieties present in each molecule (e.g., an antibody or a heterodimeric fragment thereof). Thus, a single binding molecule can bind to more than one binding site on a target antigen. Examples of multivalent antibodies include, but are not limited to, bivalent, trivalent, tetravalent, pentavalent, etc., but at least include antibodies with hexavalent valence.

[0088] As used herein, the term "multispecific" (e.g., a multispecific antibody or a heterodimeric fragment thereof) refers to a single molecule that binds to two or more different epitopes on at least two or more different antigens. The term "multispecific antibody" includes, but is not limited to, bispecific antibodies, triple specific antibodies, tetraspecific antibodies, etc. In some respects, the term "multispecific antibody" refers to a bispecific antibody. In some respects, the term "multispecific antibody" refers to a triple specific antibody. In some respects, the term "multispecific antibody" refers to an antibody with a 4, 5, or 6 valence. In some respects, the term "multispecific antibody" refers to an antibody with a valence greater than 6.

[0089] In some respects, the heterodimeric protein produced by the methods described herein is a heterodimeric IgG antibody, wherein the antibody is multivalent and / or multispecific.

[0090] In some respects, heterodimeric proteins (such as IgG antibodies or their heterodimeric fragments) may contain two or more variable regions. In some respects, each of those variable regions can specifically bind to a different epitope. In some respects, different epitopes are located on different antigens. In some respects, different antigens are expressed on the same or different cells.

[0091] In some respects, heterodimeric antibodies can contain two distinct light chains. Alternatively, heterodimeric antibodies can contain two identical light chains.

[0092] In one aspect, the heterodimeric protein produced by the method of this disclosure is an IgG antibody, which may comprise two monomers of the same IgG subclass, for example, it may comprise two IgG1 monomers, two IgG2 monomers, two IgG3 monomers, or two IgG4 monomers.

[0093] In some respects, the heterodimeric proteins produced by this disclosure (such as IgG antibodies or heterodimeric IgG fragments thereof) may comprise two identical light chains. Alternatively, the heterodimeric proteins produced by this disclosure (such as IgG antibodies or heterodimeric IgG fragments thereof) may comprise two distinct light chains.

[0094] In some respects, this document provides a method for generating a DEKK heterodimer protein comprising two distinct IgG CH3 domains capable of forming a CH3-CH3 interface, the method comprising the following steps: - supply: (a) A first protein comprising a first CH3 domain, wherein the first CH3 domain contains positively charged amino acid residues at positions 351 and 366, wherein the positively charged amino acid residues at positions 351 and 366 are K and K, respectively. (b) A second protein containing a second CH3 domain, wherein the second CH3 domain contains negatively charged amino acid residues at positions 351 and 368, wherein the negatively charged amino acid residues at positions 351 and 368 are D and E, respectively. The numbering is based on the EU numbering. - Proteins (a) and (b) are incubated together under reducing conditions to provide a first protein and a second protein that have been reduced; and - The reduced first and second proteins are re-oxidized to obtain DEKK heterodimer protein.

[0095] In some respects, this document provides a method for generating a heterodimeric antibody comprising two distinct IgG CH3 domains capable of forming a CH3-CH3 interface, the method comprising the following steps: - supply: (a) A first antibody or half, wherein each CH3 domain contains positively charged amino acid residues at positions 351 and 366, and (b) A second antibody or half-body, wherein each CH3 domain contains negatively charged amino acid residues at positions 351 and 368. The numbering is based on the EU numbering. - Incubate (a) and (b) antibodies and / or halves together under reducing conditions to provide a first antibody or halves and a second antibody or halves for reduction; and - The reduced first antibody and / or second half are re-oxidized to obtain a heterodimeric antibody.

[0096] In some respects, this document provides a method for generating IgG heterodimeric antibodies comprising two distinct IgG CH3 domains capable of forming a CH3-CH3 interface, the method comprising the following steps: - supply: (a) A first IgG antibody or IgG half, wherein each CH3 domain contains positively charged amino acid residues at positions 351 and 366, and (b) A second IgG antibody or IgG half, wherein each CH3 domain contains negatively charged amino acid residues at positions 351 and 368. The numbering is based on the EU numbering. - Incubate (a) and (b) IgG antibodies and / or IgG halves together under reducing conditions to provide a reduced first IgG antibody or IgG halves and a reduced second IgG antibody or IgG halves; and - The reduced IgG antibody and / or IgG half-body are re-oxidized to obtain heterodimeric IgG antibody.

[0097] In some respects, this document provides a method for generating DEKK heterodimeric antibodies comprising two distinct IgG CH3 domains capable of forming a CH3-CH3 interface, the method comprising the following steps: - supply: (a) A first antibody or half, wherein each CH3 domain contains positively charged amino acid residues at positions 351 and 366, wherein the positively charged amino acid residues at positions 351 and 366 are K and K, respectively. (b) A second antibody or half-body, wherein each CH3 domain contains negatively charged amino acid residues at positions 351 and 368, wherein the negatively charged amino acid residues at positions 351 and 368 are D and E, respectively. The numbering is based on the EU numbering. - Incubate (a) and (b) antibodies and / or halves together under reducing conditions to provide a first antibody or halves and a second antibody or halves for reduction; and - The reduced antibody and / or hemisome is re-oxidized to obtain DEKK heterodimer antibody.

[0098] In some respects, this document provides a method for generating DEKK heterodimeric IgG antibodies, the DEKK heterodimeric IgG antibodies comprising two distinct IgG CH3 domains capable of forming a CH3-CH3 interface, the method comprising the following steps: - supply: (a) A first IgG antibody or IgG half, wherein each CH3 domain contains positively charged amino acid residues at positions 351 and 366, wherein the positively charged amino acid residues at positions 351 and 366 are K and K, respectively. (b) A second IgG antibody or IgG half, wherein each CH3 domain contains negatively charged amino acid residues at positions 351 and 368, wherein the negatively charged amino acid residues at positions 351 and 368 are D and E, respectively. The numbering is based on the EU numbering. - Incubate (a) and (b) IgG antibodies and / or IgG halves together under reducing conditions to provide a reduced first IgG antibody or IgG halves and a reduced second IgG antibody or IgG halves; and - The reduced IgG antibody and / or IgG half are re-oxidized to obtain DEKK heterodimer IgG antibody.

[0099] Methods for preparing heterodimeric proteins

[0100] The method disclosed herein includes the steps of providing: (a) a first protein comprising a CH3 domain containing positively charged amino acid residues at positions 351 and 366 (also referred herein as the 351 / 366 positively charged CH3 domain), and (b) a second protein comprising a CH3 domain containing negatively charged amino acid residues at positions 351 and 368 (also referred herein as the 351 / 368 negatively charged CH3 domain), wherein the numbering is in accordance with EU designations.

[0101] In some respects, the first and / or second protein may comprise or be selected from: monomeric proteins, homodimeric proteins, and heterodimeric proteins.

[0102] Those skilled in the art will understand that the terms “monomer protein” and “monomer”, used interchangeably herein, generally refer to a single, non-aggregated protein or polypeptide molecule. However, as noted above, in the antibody field, the term monomer can also refer to a half-molecule. For example, an IgG monomer is an IgG half-molecule, i.e., a molecule comprising a single IgG heavy chain linked to a single IgG light chain or consisting of a single IgG heavy chain linked to a single IgG light chain. The light chain and the heavy chain are linked by disulfide bonds in the IgG half-molecule. Therefore, in the context of this disclosure, depending on the context, the term monomer refers to a single, non-aggregated protein or polypeptide molecule (such as a single IgG CH3 domain or a monomeric IgG Fc region), or a half-molecule (e.g., an IgG half-molecule).

[0103] Similarly, in the art, the term "homodimeric protein" or "homodimer" generally refers to a dimer formed by two identical polypeptides covalently or nonvalently linked (e.g., two 351 / 368 negatively charged CH3 domains or two 351 / 366 positively charged CH3 domains). However, in the field of antibodies, the term homodimer can also refer to an antibody having two identical hemispheres (such as an IgG antibody). Therefore, in the context of this disclosure, the term homodimer, depending on the context, refers to a dimer formed by two identical polypeptides covalently or nonvalently linked (e.g., two identical IgG CH3 domains), or a homodimeric antibody (e.g., a homodimeric IgG antibody).

[0104] Therefore, when the first protein is a homodimer, the method of this disclosure includes the step of providing: (a) a first protein (e.g., a homodimer IgG antibody or a fragment thereof) comprising two (identical) CH3 domains, each domain containing positively charged amino acid residues at positions 351 and 366 (also referred herein as the 351 / 366 positively charged CH3 domain). Similarly, when the second protein is a homodimer, the method of this disclosure includes the step of providing: (b) a second protein (e.g., a homodimer IgG antibody or a fragment thereof) comprising two (identical) CH3 domains, each domain containing negatively charged amino acid residues at positions 351 and 368 (also referred herein as the 351 / 368 negatively charged CH3 domain).

[0105] As described elsewhere herein, the term "heterodimeric protein" or "heterodimer" refers to a protein comprising two monomers having different polypeptides covalently or non-covalently linked. In the context of the first and second proteins described herein, it refers to a protein comprising two different monomers (i.e., having different polypeptide sequences) but comprising IgG CH3 domains having the same charge (i.e., CH3 domains in the first protein heterodimer are all 351 / 366 positively charged IgG CH3 domains, while CH3 domains in the second protein heterodimer are all 351 / 368 negatively charged IgG CH3 domains). Therefore, when the first protein is a heterodimer, the method described in this disclosure comprises the step of providing: (a) a first protein (e.g., a heterodimeric IgG antibody or a fragment thereof) comprising two CH3 domains, the CH3 domains containing positively charged amino acid residues at positions 351 and 366 (also referred to herein as 351 / 366 positively charged CH3 domains). Similarly, when the second protein is a heterodimer, the method of this disclosure includes the step of providing: (b) a second protein (e.g., a heterodimer IgG antibody or a fragment thereof) comprising two CH3 domains, wherein the CH3 domains contain negatively charged amino acid residues at positions 351 and 368, respectively (also referred to herein as the 351 / 368 negatively charged CH3 domains).

[0106] In some respects, the two IgG CH3 domains of the two monomers forming the heterodimer of the first protein have the same amino acid at positions 351 and 366, and similarly, the two IgG CH3 domains of the two monomers forming the heterodimer of the second protein have the same amino acid at positions 351 and 368. In some respects, the IgG CH3 domains of the two monomers forming the heterodimer are identical (i.e., the polypeptide sequence of the IgG CH3 domain is identical along its entire length).

[0107] In the field of antibodies, the term heterodimer can also refer to antibodies that contain or are composed of different hemispheres (such as IgG antibodies). Therefore, in the context of this disclosure, the term heterodimeric protein can also refer to a heterodimeric antibody. Such a heterodimeric antibody is prepared from two distinct hemispheres, wherein the two hemispheres contain IgG CH3 domains with the same charge (i.e., 351 / 366 positively charged or 351 / 368 negatively charged IgG CH3 domains). In some respects, the IgG CH3 domains of the two hemispheres forming the heterodimeric antibody have the same amino acid at positions 351 and 366 or positions 351 and 368 (depending on whether the IgG CH3 domain is 351 / 366 positively charged or 351 / 368 negatively charged). In some respects, the IgG CH3 domains of the two hemispheres forming the heterodimeric antibody are identical (i.e., the polypeptide sequence of the IgG CH3 domain is identical along its entire length).

[0108] In some aspects, the first and second proteins provided in methods (a) and (b) of this disclosure are monomers (e.g., the first and second proteins are first and second halves, respectively). In some aspects, the first protein (e.g., the first half) may contain a 351 / 366 positively charged IgG CH3 domain, and the second protein (e.g., the second half) may contain a 351 / 368 negatively charged IgG CH3 domain. After completing the reduction and re-oxidation steps of the methods of this disclosure, the resulting heterodimeric protein (e.g., a heterodimeric antibody) will contain the first and second proteins (e.g., the first and second halves).

[0109] In another example, the first protein provided in (a) of the method described in this disclosure is a monomer (e.g., the first protein is a half-monomer), and the second protein provided as (b) is a homodimeric protein (e.g., the second protein is a homodimeric antibody). The monomeric protein (e.g., the half-monomer) may contain a 351 / 366 positively charged IgG CH3 domain, while the homodimeric protein (e.g., the homodimeric antibody) may contain two 351 / 368 negatively charged IgG CH3 domains. After completing steps ii) and iii) of the method described in this disclosure, the resulting heterodimeric protein (e.g., the heterodimeric antibody) will contain a monomeric protein (which is a half-monomer) and one of the monomers of the homodimeric protein (e.g., one of the half-monomers of the homodimeric antibody). Therefore, the resulting heterodimeric protein (e.g., the heterodimeric antibody) will contain a 351 / 366 positively charged IgG CH3 domain and a 351 / 368 negatively charged IgG CH3 domain.

[0110] In another example, the first protein provided in (a) of the method described in this disclosure is a homodimeric protein (e.g., the first protein is a homodimeric antibody), and the second protein provided in (b) is a monomer (e.g., the second protein is a hemisome). The homodimeric protein (e.g., the homodimeric antibody) may contain two 351 / 366 positively charged IgG CH3 domains, and the monomeric protein (e.g., the hemisome) may contain a 351 / 368 negatively charged IgG CH3 domain. After completing steps ii) and iii) of the method described in this disclosure, the resulting heterodimeric protein (e.g., the heterodimeric antibody) will contain one of the monomers of the homodimeric protein (e.g., one of the hemisomes of the homodimeric antibody) and the monomeric protein (which is a hemisome). Therefore, the resulting heterodimeric protein (e.g., the heterodimeric antibody) will contain a 351 / 366 positively charged IgG CH3 domain and a 351 / 368 negatively charged IgG CH3 domain.

[0111] In another example, the first protein provided in (a) of the method described in this disclosure is a homodimeric protein (e.g., a first homodimeric antibody), and the second protein provided in (b) may also be a homodimeric protein (e.g., a second homodimeric antibody). The first homodimeric protein (e.g., the first homodimeric antibody) may contain two 351 / 366 positively charged IgG CH3 domains, and the second protein (e.g., the second homodimeric antibody) may contain two 351 / 368 negatively charged IgG CH3 domains. After completing the reduction and re-oxidation steps of the method described in this disclosure, the resulting heterodimeric protein (e.g., the heterodimeric antibody) will contain one of the monomers of the first homodimeric protein (e.g., one half of the first homodimeric antibody) and one of the monomers of the second homodimeric protein (e.g., one half of the second homodimeric antibody). Therefore, the resulting heterodimeric protein (e.g., a heterodimeric antibody) will contain a 351 / 366 positively charged IgG CH3 domain and a 351 / 368 negatively charged IgG CH3 domain.

[0112] In another example, the first protein provided in (a) of the method described in this disclosure is a monomer (e.g., the first protein is a half-body), and the second protein provided in (b) is a heterodimeric protein (e.g., the second protein is a heterodimeric antibody). The monomeric protein (e.g., the half-body) may contain a 351 / 366 positively charged IgG CH3 domain, while the heterodimeric protein (e.g., the heterodimeric antibody) may contain a 351 / 368 negatively charged IgG CH3 domain. After completing the reduction and re-oxidation steps of the method of this disclosure, the resulting heterodimeric protein (e.g., the heterodimeric antibody) will contain a monomeric protein (which is a half-body) and one of the monomers of the heterodimeric protein (e.g., one of the halves of a homodimeric antibody). Therefore, the resulting heterodimeric protein (e.g., the heterodimeric antibody) will contain a 351 / 366 positively charged IgG CH3 domain and a 351 / 368 negatively charged IgG CH3 domain.

[0113] In another example, the first protein provided in (a) of the method described in this disclosure is a heterodimeric protein (e.g., the first protein is a heterodimeric antibody), and the second protein provided in (b) is a monomer (e.g., the second protein is a hemisome). The heterodimeric protein (e.g., the heterodimeric antibody) may contain two 351 / 366 positively charged IgG CH3 domains, and the monomeric protein (e.g., the hemisome) may contain a 351 / 368 negatively charged IgG CH3 domain. After completing the reduction and re-oxidation steps of the method described in this disclosure, the resulting heterodimeric protein (e.g., the heterodimeric antibody) will contain one of the monomers of the heterodimeric protein (e.g., one of the hemisomes of the heterodimeric antibody) and the monomeric protein (which is a hemisome). Therefore, the resulting heterodimeric protein (e.g., the heterodimeric antibody) will contain a 351 / 366 positively charged IgG CH3 domain and a 351 / 368 negatively charged IgG CH3 domain.

[0114] In another example, the first protein provided in (a) of the method described in this disclosure is a heterodimeric protein (e.g., a first heterodimeric antibody), and the second protein provided in (b) may also be a homodimeric protein (e.g., a second homodimeric antibody). The first heterodimeric protein (e.g., the first heterodimeric antibody) may contain two 351 / 366 positively charged IgG CH3 domains, while the second protein (e.g., the second homodimeric antibody) may contain two 351 / 368 negatively charged IgG CH3 domains. After completing the reduction and re-oxidation steps of the method described in this disclosure, the resulting heterodimeric protein (e.g., the heterodimeric antibody) will contain one monomer of the first heterodimeric protein (e.g., one half of the first heterodimeric antibody) and one monomer of the second homodimeric protein (e.g., one half of the second homodimeric antibody). Therefore, the resulting heterodimeric protein (e.g., a heterodimeric antibody) will contain a 351 / 366 positively charged IgG CH3 domain and a 351 / 368 negatively charged CH3 domain.

[0115] In another example, the first protein provided in (a) of the method described in this disclosure is a homodimeric protein (e.g., a first homodimeric antibody), and the second protein provided in (b) may also be a heterodimeric protein (e.g., a second heterodimeric antibody). The first homodimeric protein (e.g., the first homodimeric antibody) may contain two 351 / 366 positively charged IgG CH3 domains, and the second protein (e.g., the second heterodimeric antibody) may contain two 351 / 368 negatively charged IgG CH3 domains. After completing the reduction and re-oxidation steps of the method described in this disclosure, the resulting heterodimeric protein (e.g., the heterodimeric antibody) will contain one of the monomers of the first homodimeric protein (e.g., one half of the first homodimeric antibody) and one of the monomers of the second heterodimeric protein (e.g., one half of the second heterodimeric antibody). Therefore, the resulting heterodimeric protein (e.g., a heterodimeric antibody) will contain a 351 / 366 positively charged IgG CH3 domain and a 351 / 368 negatively charged CH3 domain.

[0116] In another example, the first protein provided in (a) of the method described in this disclosure is a heterodimeric protein (e.g., a first heterodimeric antibody), and the second protein provided in (b) may also be a heterodimeric protein (e.g., a second heterodimeric antibody). The first heterodimeric protein (e.g., the first heterodimeric antibody) may contain two 351 / 366 positively charged IgG CH3 domains, and the second protein (e.g., the second heterodimeric antibody) may contain two 351 / 368 negatively charged IgG CH3 domains. After completing the reduction and re-oxidation steps of the method described in this disclosure, the resulting heterodimeric protein (e.g., the heterodimeric antibody) will contain one of the monomers of the first heterodimeric protein (e.g., one half of the first heterodimeric antibody) and one of the monomers of the second heterodimeric protein (e.g., one half of the second heterodimeric antibody). Therefore, the resulting heterodimeric protein (e.g., a heterodimeric antibody) will contain a 351 / 366 positively charged IgG CH3 domain and a 351 / 368 negatively charged CH3 domain.

[0117] In some respects, the first and / or second proteins provided in method (a) are aspects of monomeric proteins, which are monomeric Fc regions.

[0118] In some respects, the first and / or second proteins provided in method (a) are dimers (heterodimers or homodimers) that are dimer Fc regions.

[0119] In some respects, the first and / or second protein may comprise or be selected from: antibodies and haptens, or fragments thereof. Antibodies, haptens, and fragments thereof are described elsewhere herein. In some respects, said fragments are monomeric or dimer Fc regions.

[0120] In some respects, the first and / or second proteins can have sequences from any source, such as mouse and human sequences. The first and / or second proteins can consist of sequences from only one source, such as fully human antibodies, or they can have sequences from more than one source, resulting in, for example, chimeric or humanized antibodies. It is desirable that the first and / or second proteins used for treatment (such as first and / or second antibodies) be as close as possible to the natural antibody of the subject to be treated (e.g., a human antibody for a human subject). Therefore, in some respects, antibodies, haptens, or fragments thereof are human antibodies, human haptens, or fragments thereof. It will be understood that the fragment must contain at least an IgG CH3 domain, a hinge region, and a Fab arm. In some respects, the Fab arm contains a variable heavy chain domain. In some respects, the Fab arm contains a variable heavy chain domain and a variable light chain. In some respects, the Fab arm contains a variable heavy chain domain and does not contain (i.e., lacks) a variable light chain.

[0121] In some respects, when the first and / or second protein is an antibody, it is a homodimeric antibody or a heterodimeric antibody.

[0122] In some respects, the first protein is an antibody with a first binding specificity, and the second protein is an antibody with a second, different binding specificity.

[0123] In some respects, the first protein may contain an antibody with binding specificity, while the second protein may contain an antibody with a different binding specificity.

[0124] As described above, the preferential binding of the 351 / 366 positively charged CH3 domain (which contains positively charged amino acid residues at positions 351 and 366) to the 351 / 368 negatively charged CH3 domain (which contains negatively charged amino acid residues at positions 351 and 368) facilitates the generation of heterodimeric proteins according to the method described herein.

[0125] In some respects, the 351 / 366 positively charged CH3 domain may contain amino acid K or R at position 351 and amino acid K or R at position 366. For example, the 351 / 366 positively charged CH3 domain may contain amino acid K at position 351 and amino acid R at position 366, or amino acid R at position 351 and amino acid K at position 366, or amino acid K at position 351 and amino acid K at position 366, or amino acid R at position 351 and amino acid R at position 366. In some respects, the 351 / 366 positively charged CH3 domain may contain amino acid K at position 351 (i.e., may contain 351K) and amino acid K at position 366 (i.e., may contain 366K). The first protein containing both of the latter 351 / 366 positively charged CH3 domains is referred to herein as the KKKK first protein. In some respects, the KKKK first protein is a homodimer or heterodimer. In some respects, the KKKK first protein is a homodimeric IgG antibody or a fragment thereof, or a heterodimeric IgG antibody or a fragment thereof. In other respects, the KKKK IgG antibody or a fragment thereof is an IgG1, IgG2, IgG3, or IgG4 antibody or a fragment thereof.

[0126] In some respects, the 351 / 368 negatively charged CH3 domain may contain amino acids D or E at position 351 and D or E at position 368. For example, the 351 / 368 negatively charged CH3 domain may contain amino acid D at position 351 and amino acid E at position 368, or amino acid E at position 351 and amino acid D at position 368, or amino acid E at position 351 and amino acid E at position 368, or amino acid D at position 351 and amino acid D at position 368. In some respects, the 351 / 368 negatively charged CH3 domain may contain amino acid D at position 351 (i.e., may contain 351D) and amino acid E at position 368 (i.e., may contain 368E). A second protein containing two of the latter 351 / 368 negatively charged CH3 domains is referred to herein as a DEDE second protein. In some respects, the DEDE second protein is a homodimer or heterodimer. In some respects, the DEDE second protein is a homodimeric IgG antibody or a fragment thereof, or a heterodimeric IgG antibody or a fragment thereof. In other respects, the DEDE IgG antibody or a fragment thereof is an IgG1, IgG2, IgG3, or IgG4 antibody or a fragment thereof.

[0127] In some respects, the 351 / 366 positively charged CH3 domain may contain amino acid K at position 351 (i.e., may contain 351K) and amino acid K at position 366 (i.e., may contain 366K), and the 351 / 368 negatively charged CH3 domain may contain amino acid D at position 351 (i.e., may contain 351D) and amino acid E at position 368 (i.e., may contain 368E). As described above, a heterodimer having one 351 / 366 positively charged CH3 domain and one 351 / 368 negatively charged CH3 domain is referred to herein as a DEKK heterodimer. In some respects, the DEKK heterodimer is a heterodimeric IgG antibody or a fragment thereof. In some respects, the DEKK IgG antibody or a fragment thereof is an IgG1, IgG2, IgG3, or IgG4 antibody or a fragment thereof.

[0128] The above amino acid positions will be understood based on the human IgG CH3 domain. However, in one aspect of utilizing the non-human IgG CH3 domain, the same amino acid substitutions are introduced into the corresponding amino acid residues.

[0129] In some aspects, the first and second proteins are provided in a ratio that is advantageous for producing heterodimeric proteins by the method described herein. "Advantageous" means that it increases the proportion of the resulting heterodimeric protein (wherein the heterodimeric protein has a 351 / 366 positively charged CH3 domain and a 351 / 368 negatively charged CH3 domain) compared to providing equal amounts of the first and second proteins. In some aspects, the ratio of the first protein (containing a 351 / 366 positively charged IgG CH3 domain) to the second protein (containing a 351 / 368 negatively charged IgG CH3 domain) is between 20:1 and 1:20 (w / w). Ratios exceeding these amounts may be used, but this may practically reduce the effective use of the protein. In some aspects, the ratio is between 10:1 and 1:10. In some aspects, the ratio is between 5:1 and 1:5. In some aspects, the ratio is at least 1:1 (w / w), for example at least 1.2:1 (w / w), for example at least 1.5:1 (w / w), or at least 2:1 (w / w). In some respects, the ratio of the first protein (containing a 351 / 366 positively charged IgG CH3 domain) to the second protein (containing a 351 / 368 negatively charged IgG CH3 domain) is between 1:1 (w / w) and 2:1 (w / w).

[0130] In some respects, the ratio of KKKK first protein (containing 351 / 366 positively charged IgG CH3 domains) to DEDE second protein (containing 351 / 368 negatively charged IgG CH3 domains) is at least 1:1 (w / w), for example at least 1.2:1 (w / w), for example at least 1.5:1 (w / w), or at least 2:1 (w / w). In some respects, the ratio of KKKK first protein (containing 351 / 366 positively charged IgG CH3 domains) to DEDE second protein (containing 351 / 368 negatively charged IgG CH3 domains) is between 1:1 (w / w) and 2:1 (w / w).

[0131] In some aspects, the ratio of KK first protein (containing a 351 / 366 positively charged IgG CH3 domain) to DEDE second protein (containing a 351 / 368 negatively charged IgG CH3 domain) is 20:1 to 1:20 (w / w). In some aspects, the ratio is between 10:1 and 1:10. In some aspects, the ratio is between 5:1 and 1:5. In some aspects, the ratio is at least 1:1 (w / w), for example at least 1.2:1 (w / w), for example at least 1.5:1 (w / w), or at least 2:1 (w / w). In some aspects, the ratio of KK first protein (containing a 351 / 366 positively charged IgG CH3 domain) to DEDE second protein (containing a 351 / 368 negatively charged IgG CH3 domain) is between 1:1 (w / w) and 2:1 (w / w).

[0132] It will be understood that when the first and second proteins are incubated under reducing conditions and then re-oxidized, any appropriate amount of the first and second proteins may be used. Those skilled in the art will determine the appropriate concentration, for example, using the methods described in the Examples section below. As a non-limiting example, each of the first and second proteins may be used at a concentration of at least 50 μg / ml, such as at least 0.1 mg / ml, at least 1.0 mg / ml, at least 10 mg / ml, or at least 25 mg / ml but not exceeding 100 mg / ml, under the reducing conditions. In some aspects, when each of the first and second proteins is an antibody, the first and second proteins may each be used at a concentration of at least 50 μg / ml, such as at least 0.1 mg / ml or at least 1.0 mg / ml, under the reducing conditions. Typically, they may each be used in the concentration range of 50 μg / ml to 2 mg / ml, for example, in the range of 50 μg / ml to mg / ml. By way of example only, they may each be used at a concentration of about 1.1 mg / ml.

[0133] It will also be understood that other reagents may be present under the reduction conditions to facilitate the process of obtaining a reduced first protein and a reduced second protein. For example, in one aspect, cystamine is added under reduction conditions at an appropriate concentration, for example, in the range of 2 to 70 mM, to further reduce any homodimers present in the reaction. For example, when the first and second proteins are antibodies (such as homodimer antibodies), cystamine is added under reduction conditions at a concentration of about 2 to about 70 mM. In other aspects, cystamine is added under reduction conditions in the range of 2 to 32 mM to further reduce any homodimers present in the reaction. For example, when the first and second proteins are antibodies (such as homodimer antibodies), cystamine is added under reduction conditions at a concentration of about 2 to about 32 mM.

[0134] In some respects, the first and second proteins are obtained independently of each other. By way of example only, the first and second proteins are produced using different host cells. Exemplary methods for obtaining the first and second proteins are provided in the following Examples section. However, other methods will be known to those skilled in the art.

[0135] "Host cell" can be any host cell known in the art that is capable of expressing recombinant DNA molecules and expressing binding moieties.

[0136] In some respects, the first protein (like a dimer antibody) and / or the second protein (like a dimer antibody) are obtained under serum-free conditions (e.g., by culturing host cells in FreeStyle 293 or FreeStyle Cho medium, Invitrogen).

[0137] In some aspects, the first and / or second proteins provided in (a) and (b) are purified using methods known in the art between incubation under the reducing conditions of the method. These methods may include precipitation, centrifugation, filtration, size exclusion chromatography, affinity chromatography, cation and / or anion exchange chromatography, hydrophobic interaction chromatography, etc. For antibody mixtures containing IgG molecules, protein A or protein G affinity chromatography may be used (see, for example, U.S. Patents 4,801,687 and 5,151,504).

[0138] The incubation under the reducing and reoxidizing conditions of this method facilitates the generation of a heterodimeric protein through recombination of the first and second proteins (such as antibodies or their dimer fragments) or the binding of the first and second proteins (such as their hemimeric or monomeric fragments). Recombination of the first and second proteins occurs when the first and second proteins exchange monomers (e.g., their hemimeric or dimeric fragments) to generate a heterodimeric protein containing two distinct IgG CH3 domains capable of forming a CH3-CH3 interface.

[0139] As used herein, the term "incubation" refers to holding, retaining, or maintaining the first and second proteins together under relevant conditions (i.e., reducing conditions). This involves incubating the first and second proteins together in a composition or formulation containing the first and second proteins.

[0140] The incubation under the reduction conditions of the method includes incubating the proteins of (a) and (b) together under reduction conditions to provide a reduced first protein and / or a reduced second protein.

[0141] The term "reducing conditions" refers to an environment in which the first and / or second protein is more likely to be reduced rather than oxidized. In some respects, reducing conditions can lead to "disulfide bond reduction" (i.e., the breaking of disulfide bonds, resulting in the formation of two thiol groups (-SH groups)). Those skilled in the art will understand that when antibodies (homodimolecular or heterodimeric antibodies) are incubated under reducing conditions, the reduction of disulfide bonds (such as those in the core hinge region) can cause the antibody to separate into two halves.

[0142] The step of incubating the first and second proteins under reducing conditions may include incubating the first and second proteins in the presence of a reducing agent. The term "reducing agent" refers to a compound that reduces molecules in its environment, i.e., a compound that alters molecules in its environment to become more reduced. A reducing agent can act by donating electrons, thereby being oxidized itself after reducing the substrate (i.e., the first and / or second protein).

[0143] Examples of reducing agents include 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, β-mercaptoethanol, mercaptoacetate, cysteamine, homocysteine, penicillamine, and / or sodium borohydride.

[0144] In some respects, when the reducing agent is 2-MEA, its concentration is from about 25 mM to about 100 mM, for example from about 50 mM to about 75 mM. In some respects, the concentration of 2-MEA is about 75 mM.

[0145] It will be understood that the incubation time with a reducing agent can depend on the concentration and / or temperature at which the incubation is performed. For example, a higher concentration of reducing agent may allow for a shorter incubation time and / or a lower incubation temperature.

[0146] In some respects, when the concentration of 2-MEA is about 75 mM, the incubation is carried out at about 31°C and lasts for at least 300 minutes.

[0147] In one respect, the reducing agent does not contain enzymes.

[0148] Alternatively, incubating the first and second proteins under reducing conditions may include incubating them at a pH of 6.0 or higher, such as at a pH of 7.0 or higher, 8.0 or higher, 9.0 or higher, 10.0 or higher, 11.0 or higher, or 12.0. For example, the first and second proteins may be incubated at a pH between 6.0 and 11.0, or between 6.0 and 10.0, optionally with a pH between 7.0 and 8.0 (e.g., between pH 7.3 and 7.5). In some respects, the pH is 7.4. Alternatively, incubating the first and second proteins under reducing conditions may include incubating the proteins at a redox potential between -150 and -600 mV, optionally with a redox potential between -250 and -400 mV. Examples of suitable reducing conditions are known in the art. Some examples are described in Labrijn AF., Nature Protocols 2014, Volume 9, Issue 10, pp. 2450-2463.

[0149] The method also includes re-oxidizing the reduced protein to obtain a heterodimeric protein.

[0150] The terms “reoxidation” or “oxidation” as used refer to a reaction in which electrons are lost to another substance, or a reaction that results in such a reaction. In the context of this disclosure, the reoxidation step can allow the resulting reduced first protein and reduced second protein to combine to form a heterodimeric protein comprising two distinct IgG CH3 domains capable of forming a CH3-CH3 interface.

[0151] Because a first protein with a 351 / 366 positively charged IgG CH3 domain and a second protein with a 351 / 368 negatively charged IgG CH3 domain preferentially bind to each other, a larger proportion of heterodimeric proteins containing two different IgG CH3 domains will be formed compared to dimeric proteins with the same charge of IgG CH3 domains (homodimer or heterodimer). Therefore, this disclosure provides a method for the efficient and controllable production of mixtures of well-defined Ig antibodies or heterodimeric fragments thereof in a high proportion of bispecificity in the mixture. In systems where bispecificity is desired, even a bispecificity ratio of at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or higher can be obtained. This means obtaining only 5% or less, or 3% or less, of monospecific divalent byproducts. Notably, the monomeric byproducts, i.e., half-molecules, produced by the methods described herein are more stable than monomeric byproducts produced by at least some other methods known in the art (such as those described in WO2011131746). This is advantageous because these half-molecules can be further subjected to reducing conditions (discussed elsewhere in this disclosure) to produce the desired heterodimeric protein without requiring repeated oxidation steps.

[0152] In some respects, the reoxidation of reduced proteins is achieved by removing the reducing agent from the mixture of the reduced first protein and the reduced second protein. By way of example only, the reducing agent is removed by percolation (e.g., as described in Labrijn AF., Nature Protocols 2014, Vol. 9, No. 10, pp. 2450-2463). It will be understood that complete removal of the reducing agent is not necessary for the reoxidation of the reduced first and second proteins. For example, in the context of 2-MEA as the reducing agent, reducing the concentration to less than 50 μM may be sufficient to reoxidize the reduced first and second proteins. Another example of a method for reoxidizing reduced proteins is buffer exchange. In some respects, the buffer exchange is performed with PBS. Optionally, the pH of the PBS is 7.4. Methods for performing buffer exchange are known to those skilled in the art. In some respects, buffer exchange is performed using Zeba plates, as illustrated in the Examples section of this disclosure. As another example, PBS can be buffer-exchanged using an ÄKA Pure 25 system with a Spark ALIAS autosampler and a desalting column (e.g., HiPrep 26 / 10). In this example, buffer exchange can occur at a flow rate of 6 mL / min and a temperature of 20 degrees Celsius.

[0153] After buffer exchange, the sample can be kept at approximately 4°C for approximately 24, 48, 64 hours or longer to allow for complete re-oxidation.

[0154] As an alternative or in addition, the re-oxidation step includes incubating the reduced first protein and the reduced second protein together with an oxidant.

[0155] In some aspects, the method further includes the steps of enriching and / or separating the obtained heterodimeric proteins. Heterodimeric proteins can be enriched and / or separated from any contaminants that may be generated by the methods described herein using conventional methods, such as conventional purification methods. These contaminants may include homodimeric proteins (proteins comprising two monomers, each having the same IgG CH3 domain) and / or monomeric proteins (e.g., hemisomes). Methods for purifying the resulting heterodimeric proteins may include precipitation, centrifugation, filtration, size exclusion chromatography, affinity chromatography, cation and / or anion exchange chromatography, hydrophobic interaction chromatography, etc. For antibody mixtures containing IgG molecules, protein A or protein G affinity chromatography may be used (see, for example, U.S. Patents 4,801,687 and 5,151,504).

[0156] In some aspects, the incubation under reducing conditions and the reoxidation of the reduced protein together are also described as incubating the first and second proteins under conditions sufficient to allow cysteine ​​residues in the CH3 region to undergo disulfide isomerization to obtain a heterodimeric protein. In some aspects, when the first and second proteins are antibodies or haptens, the incubation under reducing conditions and the reoxidation of the reduced protein together are described as incubating the first and second proteins under conditions sufficient to allow cysteine ​​residues in the core hinge region of the first and second proteins to undergo disulfide isomerization to obtain a heterodimeric protein.

[0157] In some aspects, the first protein and / or the second protein comprises a fusion protein. The fusion protein may contain an antibody-binding domain, an scFv, a ligand, a protein receptor, or a cytokine. In some aspects, the heterodimeric protein is a bifunctional or multifunctional fusion protein.

[0158] In another respect, this document provides isolated heterodimeric proteins that are obtainable by the methods of this disclosure.

[0159] In some respects, the heterodimeric protein that can be obtained by the methods described in this disclosure is an IgG antibody, such as IgG1, IgG2, IgG3 or IgG4.

[0160] In some respects, IgG antibodies may contain two light chains with different sequences.

[0161] On the other hand, this article provides isolated heterodimeric antibodies comprising a 351 / 366 positively charged IgG CH3 domain and a 351 / 368 negatively charged IgG CH3 domain, wherein the 351 / 366 positively charged CH3 domain and the 351 / 368 negatively charged CH3 domain are capable of forming a CH3-CH3 interface, and wherein the 351 / 368 negatively charged CH3 domain comprises one or more of amino acid variants 351E, 351D, 368E, or 368D, and the 351 / 366 positively charged CH3 domain comprises one or more of amino acid variants 366K, 366R, 351K, or 351R, and the heterodimeric antibody further comprises two light chains with different sequences.

[0162] In some respects, the 351 / 368 negatively charged CH3 domain contains 351D and 368E, and the 351 / 366 positively charged IgGCH3 domain contains 366K and 351K.

[0163] It will be understood that, in the context of the methods described herein, the aspects relating to heterodimeric proteins mentioned herein also apply to heterodimeric proteins obtainable by the methods described herein, and to isolated heterodimeric antibodies comprising a 351 / 366 positively charged IgG CH3 domain and a 351 / 368 negatively charged IgG CH3 domain, wherein the 351 / 366 positively charged CH3 domain and the 351 / 368 negatively charged CH3 domain are capable of forming a CH3-CH3 interface, wherein the 351 / 368 negatively charged CH3 domain comprises one or more of amino acid variants 351E, 351D, 368E, or 368D, and the 351 / 366 positively charged CH3 domain comprises one or more of amino acid variants 366K, 366R, 351K, or 351R, and the heterodimeric antibody further comprises two light chains having different sequences.

[0164] The changes at positions 351 / 366 and / or 351 / 368 may be combined with any of the modifications mentioned in WO2020 / 226502 A2, the entire contents of which are incorporated herein by reference. Similarly, the changes at positions 351 / 366 and / or 351 / 368 may be combined with any of the modifications mentioned in WO2021 / 235936 A1, the entire contents of which are incorporated herein by reference.

[0165] In another aspect, this document provides pharmaceutical compositions comprising the isolated heterodimeric protein of this disclosure and a pharmaceutically acceptable carrier. The term "pharmaceutical composition" refers to a formulation in which the contained active ingredient (e.g., the heterodimeric protein of this disclosure) is bioavailable and free of other components that would have unacceptable toxicity to a subject administering the formulation. The term "pharmaceuticalally acceptable carrier" refers to any carrier useful for the dissolution of the reagent (e.g., the heterodimeric protein of this disclosure) and delivery to a subject. A variety of pharmaceutically acceptable carriers are known in the art. By way of example only, these include saline, phosphate-buffered saline, or phosphate-buffered saline. The compositions may also routinely contain pharmaceutically acceptable concentrations of salt, buffers, preservatives, compatible carriers, supplemental immunostimulants such as adjuvants and cytokines, and optionally other therapeutic agents. The compositions may also include antioxidants and / or preservatives. As antioxidants, thiols (e.g., thioglycerol, cysteine, acetylcysteine, cystine, dithioerythritol, dithiothreitol, glutathione), tocopherol, butylated hydroxyanisole, butylated hydroxytoluene, sulfites (e.g., sodium sulfate, sodium bisulfite, sodium acetone bisulfite, sodium metabisulfite, sodium sulfite, sodium formaldehyde sulfoxylate, sodium thiosulfate), and nordihydroguaiac acid can be mentioned. Suitable preservatives can be, for example, phenol, chlorobutanol, benzyl alcohol, methylparaben, propylparaben, benzalkonium chloride, and hexadecylpyridine chloride.

[0166] Terms and Conditions

[0167] 1. A method for generating a heterodimeric protein, said heterodimeric protein comprising two distinct IgG CH3 domains capable of forming a CH3-CH3 interface, said method comprising the following steps: - supply: (a) A first protein comprising a first CH3 domain, wherein the first CH3 domain contains positively charged amino acid residues at positions 351 and 366, and (b) A second protein containing a second CH3 domain, wherein the second CH3 domain contains negatively charged amino acid residues at positions 351 and 368. The numbering is based on the EU numbering. - Proteins (a) and (b) are incubated together under reducing conditions to provide a first protein and a second protein that have been reduced; and - The reduced first and second proteins are re-oxidized to obtain heterodimeric proteins.

[0168] 2. The method according to Clause 1, wherein the first protein and / or the second protein comprises or is selected from: monomeric proteins, homodimeric proteins, and heterodimeric proteins.

[0169] 3. The method according to any one of the preceding clauses, wherein the IgG CH3 domain is an IgG1, IgG2, IgG3 or IgG4 CH3 domain, optionally wherein the CH3 domain is a human CH3 domain.

[0170] 4. The method according to any one of the preceding clauses, wherein the first protein and / or the second protein comprises or is selected from: antibodies and haptens or fragments thereof, optionally wherein the antibody, hapten or fragment thereof is a human antibody, hapten or fragment thereof.

[0171] 5. The method according to Clause 4, wherein the segment is a monomeric Fc region containing a hinge or a dimer Fc region containing a hinge.

[0172] 6. The method according to clause 4 or 5, wherein the antibody, hapten, or fragment thereof is an IgG1, IgG2, IgG3, or IgG4 antibody, hapten, or fragment thereof.

[0173] 7. The method according to any one of the preceding clauses, wherein the first protein is an antibody having a first binding specificity, and the second protein is an antibody having a second different binding specificity.

[0174] 8. The method according to any one of the preceding clauses, wherein the first protein and / or the second protein is a homodimeric antibody.

[0175] 9. The method according to any one of the preceding clauses, wherein the first protein and / or the second protein is a heterodimeric antibody.

[0176] 10. The method according to any one of the preceding clauses, wherein the heterodimeric protein obtained by re-oxidizing the reduced protein is a heterodimeric antibody.

[0177] 11. The method according to Clause 9 or 10, wherein the heterodimeric antibody is multivalent, optionally wherein the multivalent antibody is a divalent, trivalent, tetravalent antibody or has up to a hexavalent antibody.

[0178] 12. The method according to any one of clauses 9 to 11, wherein the heterodimeric antibody is a multispecific antibody, optionally wherein the multispecific antibody is a bispecific, trispecific, or tetraspecific antibody.

[0179] 13. The method according to any one of clauses 9 to 12, wherein the heterodimeric antibody comprises two distinct light chains.

[0180] 14. The method according to any one of clauses 9 to 12, wherein the heterodimeric antibody comprises a single light chain.

[0181] 15. The method according to any one of clauses 9 to 12, wherein the heterodimeric antibody comprises three light chains.

[0182] 16. The method according to any one of clauses 9 to 12, wherein the heterodimeric antibody comprises three different light chains.

[0183] 17. The method according to any one of clauses 9 to 12, wherein the heterodimeric antibody comprises two identical light chains and another light chain different from the two identical light chains.

[0184] 18. The method according to any one of the preceding clauses, wherein the light chain is any member of the κ or λ family.

[0185] 19. The method according to any one of the preceding clauses, wherein the first protein and the second protein contain the same hinge region.

[0186] 20. The method according to any one of the preceding clauses, wherein in some respects, the first protein and the second protein comprise an IgG1 hinge region.

[0187] 21. The method according to any one of the preceding clauses, wherein the first protein and the second protein comprise an IgG2 hinge region.

[0188] 22. The method according to any one of the preceding clauses, wherein the first protein and the second protein comprise an IgG3 hinge region.

[0189] 23. The method according to any one of the preceding clauses, wherein the first protein and the second protein comprise the IgG4 hinge region.

[0190] 24. The method according to any one of the preceding clauses, wherein the first CH3 structural domain comprises: 351K and 366R, 351R and 366K, 351K and 366K, or 351R and 366R.

[0191] 25. The method according to any one of the preceding clauses, wherein the second CH3 structural domain comprises: 351D and 368E, 351E and 368D, 351D and 368D, or 351E and 368E.

[0192] 26. The method according to any one of the preceding clauses, wherein the first CH3 structural domain comprises 351K and 366K, and the second CH3 structural domain comprises 351D and 368E.

[0193] 27. The method according to any one of the preceding clauses, wherein the first and / or second protein of a) and b) comprises one or more Fc modifications.

[0194] 28. The method according to any one of the preceding clauses, wherein the first and / or second proteins of a) and b) are Fc-engineered proteins.

[0195] 29. The method according to any one of the preceding clauses, wherein the first and / or second protein in a) and b) is an Fc-silencing or Fc-enhancing protein.

[0196] 30. The method according to any one of the preceding clauses, wherein the first and / or second protein of a) and b) comprises a CH2 domain having a mutation affecting ADCC at position 235 and / or 236.

[0197] 31. The method according to any one of the preceding clauses, wherein the second protein is obtained independently of the first protein.

[0198] 32. The method according to any one of the preceding clauses, wherein the reduction conditions include: (a) Incubating the protein in the presence of a reducing agent, optionally wherein the reducing agent comprises or is selected from: 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, β-mercaptoethanol, thioglycolate, cysteamine, homocysteine, penicillamine, and sodium borohydride; and / or (b) Incubate the protein at a pH between 6.0 and 12.0, optionally wherein the pH is between 7.0 and 8.0; and / or (c) The protein is incubated at a redox potential between -150 and -600 mV, optionally wherein the redox potential is between -250 and -400 mV.

[0199] 33. The method according to any one of the preceding clauses, wherein the method further comprises the steps of enriching and / or separating the heterodimeric protein obtained after reoxidizing the reduced protein.

[0200] 34. The method according to Clause 33, wherein the heterodimeric protein is enriched and / or separated using methods including or selected from: precipitation, centrifugation, filtration, size exclusion chromatography, affinity chromatography, cation and / or anion exchange chromatography and hydrophobic interaction chromatography.

[0201] 35. An isolated heterodimeric protein, which can be obtained by any of the methods described in any of the preceding clauses.

[0202] 36. An isolated heterodimeric antibody comprising a first IgG CH3 domain and a second IgG CH3 domain, wherein the first CH3 domain and the second CH3 domain are capable of forming a CH3-CH3 interface. The first CH3 domain comprises one or more amino acid variants 366K, 366R, 351K, or 351R, and The second CH3 domain comprises one or more amino acid variants 351E, 351D, 368E, or 368D. The heterodimeric antibody also contains two or more light chains with different sequences.

[0203] 37. The isolated heterodimeric antibody according to Clause 36, wherein the first CH3 domain comprises 351K and 366K, and the second CH3 domain comprises 351D and 368E.

[0204] 38. A pharmaceutical composition comprising a heterodimeric protein isolated according to clause 35, or a heterodimeric antibody isolated according to clause 36 or 37, and a pharmaceutically acceptable carrier.

[0205] 39. The method according to any one of clauses 1-34, wherein the first protein and / or the second protein comprises an antibody-binding domain, scFv, a ligand, a protein receptor, or a cytokine.

[0206] 40. The method according to Clause 39, wherein the heterodimeric protein is a bifunctional or multifunctional fusion protein.

[0207] Example

[0208] Example 1: Transfection, expression, and purification of IgG antibodies for Fab arm exchange (FAE)

[0209] The purpose of the following experiments is to directly compare the results of FAE using the different variants listed in Table 1 (i.e., DEKK variant vs. variant 405L-409R).

[0210] Using the ExpiFectamine™ 293 Transfection Kit (ThermoFisher Scientific, catalog number #A14635) and OptiMEM I serum-depleted medium (Gibco, catalog number #31985062), Expi293F™ cells (ThermoFisher Scientific) cultured in 100 mL of Expi293F™ expression medium (ThermoFisher Scientific, catalog number #A14351010) were transiently transfected with various expression vectors encoding heavy chains (HC) and light chains (LC) (see Table 1).

[0211] Table 1. Expression vectors used to generate FAE antibodies

[0212] Table 1: Expression vectors (#1-8) were used for transfection and generation of HC and LC molecules for final IgG production. The expression vectors contained a DNA construct encoding a pattern heavy chain (HC) and a pattern light chain (LC) with the CH3 domain having the modifications shown.

[0213] In short, prepare the DNA-Opti-MEM mixture and the ExpiFectamine-Opti-MEM mixture by diluting 50 μg of plasmid DNA (0.1 mL from a 0.5 mg / mL DNA stock solution) with 3 mL of Opti-MEM™ I medium by vortexing or inverting, and diluting 0.16 mL of ExpiFectamine™ 293 reagent with 2.8 mL of Opti-MEM, then incubating for 5 minutes. Next, add the DNA-Opti-MEM mixture to the ExpiFectamine-Opti-MEM mixture, invert the tube 4-5 times, and incubate at room temperature for 15 minutes. Then, drop the full volume of ExpiFectamine™ 293 / plasmid DNA complex (approximately 6.06 mL) into the cells, gently vortexing the flask during addition, and then incubate (37°C incubator, ≥80% relative humidity and 8% CO2, at 155 rpm on an orbital shaker).

[0214] 18–22 hours post-transfection, add Enhancer 1 and Enhancer 2 from the ExpiFectamine™ 293 Transfection Kit to the transfection bottle while gently vortexing. On day 6 post-transfection, collect the culture medium and centrifuge the cells at 500g for 10 minutes at RT. Collect the supernatant and transfer it to a new 50ml tube, then centrifuge the cells at 3000g for 20 minutes. Filter the supernatant using a 0.45μm filter at the top of the bottle and measure IgG concentration using the ForteBIO Octet-QK system based on biomembrane interference (BLI) technology. This allows for real-time quantification and kinetic characterization of biomolecular interactions. Use the supernatant for ÄKTA purification.

[0215] Culture supernatant containing 9–12 mg of IgG was purified using a Protein A column (GE Healthcare / Product Catalog No. #11-0034-95, according to GE Healthcare instructions) using the ÄKTA pure system (Cytiva, EN490, Serial No.: 2031829), eluted in 0.1 M citrate buffer (pH 3.0), and immediately neutralized in an equal volume of 1.0 M Tris-HCl (pH 8.0), or directly reburied in PBS using a desalting column (Cytiva #17-1408-01). The purified IgG molecules were used in the FAE as described in Example 2.

[0216] According to the Lambert-Beer law, PBS was used as a blank for correction, and a universal molar extinction coefficient of 1.45 mol was used. -1 dm 3 cm -1 The amino acid composition of all samples was adjusted, and the concentration of all samples was determined by measuring the absorbance of the protein solution at 280 nm. Antibody concentrations ranged from 1.2 to 2.1 mg / mL and were prepared in PBS at pH 7.4.

[0217] Example 2: FAE Solution

[0218] Briefly, IgG molecules were incubated at 31°C, pH 7.4, in the presence of 75 mM MEA (β-mercaptoethylamine hydrochloride) without shaking. After 5 hours, the sample buffer was replaced with PBS pH 7.4 (Bex) using a Zeba plate at room temperature. For re-oxidation, the samples were kept at 4°C for at least one night.

[0219] The IgG molecules produced in Example 1 were subjected to a FAE reaction in a 96-well configuration, as shown in Table 2.

[0220] Table 2. Expressed IgG antibodies used in FAE

[0221] Table 2. IgG (half) antibodies expressing the CH3 modification shown. FAE reactions #1-4 consist of a mixture of two IgG molecules, while FAE reactions #5-12 consist of a single IgG as a control.

[0222] A fresh MEA stock solution (750 mM) was prepared by dissolving 852 mg cysteine ​​hydrochloride (Sigma product catalog number #30078) in 5–6 mL PBS (pH 7.4). The pH was adjusted to 7.3–7.5 by adding NaOH (5 M) to the solution at room temperature, and the solution was filled with PBS at pH 7.4 until a final volume of 10 mL was achieved to obtain a 750 mM stock solution. The solution was filtered through a 0.2 μm filter before use.

[0223] In short, IgG samples as listed in Table 2 were prepared in a deep-well plate (plate 1, 175 μL final volume adjusted to pH 7.4 with PBS) using IgG at a concentration of 1.1 mg / mL. In another deep-well plate (plate 2), 11 μL of reducing agent 750 mM MEA was pipetted into each of the 12 wells. Each 100 μL sample prepared in plate 1 was gently mixed with the MEA in plate 2, sealed with an aluminum seal, and incubated at 31°C for 5 hours without shaking. The remaining material (75 μL) in plate 1 was used as a non-reacting control, i.e., not exposed to MEA or Bex, and stored at 4°C protected from light until further use. After incubation, 100 μL of each sample was used for buffer exchange (BEX) using a Zeba™ spin desalting plate, 96 wells (Thermo Fisher Scientific, catalog number #89807).

[0224] Buffer exchange: The sample was buffer-exchanged with PBS pH 7.4 (1×, Gibco catalog #10010-015) using the Zeba™ Rotary Desalting Kit (Thermo Fisher Scientific, catalog #89807) to remove reducing agents and allow disulfide bonds to reform.

[0225] In short, Zeba TM Rotate the desalting plate to equilibrate to room temperature and assemble it on top of the wash plate. Centrifuge the plate assembly to remove the storage solution and blot the wash plate dry on a paper towel. Perform three wash steps by adding washing buffer (1×, Gibco catalog number #10010-015), then centrifuge the plate, discard the runoff, and blot dry on a paper towel.

[0226] Then, Zeba TMDesalting plates are stacked on top of the collection plate, and IgG samples are loaded into the wells and centrifuged. The flow-through containing IgG is transferred to another Zeba plate. TM Plate and centrifuge. For a more thorough buffer exchange, transfer the flow-through to another Zeba plate. TM Plate and centrifuge. All centrifugations were performed at 1000×g for 2 minutes. The mixture will be passed through Zeba... TM The third flow-through containing the sample from the three plates was retained for IgG concentration measurement. The buffer-exchanged sample was stored in a covered collection plate and kept overnight at 4°C (without further pipetting, shaking, or mixing) to allow for complete re-oxidation of the material. The buffer-exchanged sample and unreacted control stored at 4°C on plate 1 were measured on a Lunatic system as described in Example 1. The obtained IgG molecules were processed using Labchip for SDS-PAGE under specified reducing and non-reducing conditions, and analyzed using CIEX and HP-SEC as appropriate.

[0227] Example 3: LabChip Analysis

[0228] Under non-reducing conditions, following the manufacturer's instructions, approximately 1 μg of purified reaction product (including non-reaction controls) of IgG was analyzed using the Protein Clear HR kit (dye solution, sample buffer, protein gel matrix, protein ladder, low molecular weight marker, washing buffer; Perkin Elmer CLS960014) and the Protein Expression Assay LabChip (GXII Touch HT; Perkin Elmer) with GXII Touch HT (Elmer760499) in LabChip, according to the Protein Clear HR kit (Dye Solution, Sample Buffer, Protein Gel Matrix, Protein Ladder, Low Molecular Weight Marker, Wash Buffer; Perkin Elmer CLS960014).

[0229] IgG, half-molecules, and IgG-1 LC bands were quantified from samples run under non-reducing conditions using Labchip RX reviewer software. Only samples in which the IgG-1 LC band constituted less than 15% of the signal in the sample undergoing FAE were analyzed. Additionally, only samples with starting materials containing more than 95% intact molecules (IgG dimers or half-molecules) were included. Figure 1 Results were provided.

[0230] Samples collected before the FAE reaction showed the following: those containing the CH3-DE variant primarily formed DEDE homodimers, while samples containing the CH3 KK variant primarily formed hemimers. Single-arm products (in production) with the CH3 variants 405L or 409R primarily formed homodimers. Samples containing mixtures of DE / KK or 405L / 409R showed the following... Figure 1The bands shown; samples from the reaction containing the DE / KK CH3 variant showed a mixture of homodimers and hemimers, while samples from proteins containing the CH3 variant 405L / 409R showed mainly homodimers.

[0231] The single product (production) of samples obtained after FAE reaction with DE or KK CH3 variants mainly showed as half-mers, while samples with 405L or 409R CH3 variants formed homodimers.

[0232] Samples containing CH3-containing DE / KK species obtained after FAE reaction showed IgG peaks with half-dimer contaminants, while samples containing 405L / 409R species showed IgG peaks with unseparated homodimer contaminants.

[0233] Example 4: HP-CIEX Analysis

[0234] To confirm that the FAE reaction indeed resulted in the production of bispecific antibodies, all samples generated in Example 2 (reaction products and unreacted controls) were analyzed using CIEX (Agilent 1260 series). The column (TSK gel SP-STAT 7μm, 4.6mm ID×10cm L; Tosoh catalog number #21964) was equilibrated with a low-ionic-strength phosphate buffer (Buffer A) (25mM sodium phosphate, pH 6.0±0.05, composed of sodium dihydrogen phosphate dihydrate (NaH2PO4,2H2O; Sigma, ref. 71500) and disodium hydrogen phosphate dihydrate (Na2HPO4,2H2O; Sigma, ref. 71643) dissolved in Milli-Q water, filtered through a 0.45μm membrane filter). Then, the antibody was displaced from the column by running a phosphate buffer gradient with gradually increasing percentages of salt-containing phosphate buffer (Buffer B) (25 mM sodium phosphate, 1 M NaCl, pH 6.0, composed of sodium dihydrogen phosphate dihydrate (NaH2PO4, 2H2O; Sigma, ref. 71500) and disodium hydrogen phosphate dihydrate (Na2HPO4, 2H2O; Sigma, ref. 71643) and NaCl (Sigma, ref. S3014) dissolved in Milli-Q water, filtered through a 0.45 μm membrane filter). All test and control samples were injected at a mass of 10 μg in volumes ranging from 10 to 100 μL. Peak shapes and retention times were analyzed in the chromatograms. The peak area of ​​the main peak was observed based on the 220 nm result.

[0235] The percentage of bispecific antibodies in the samples subjected to FAE was used as the percentage of the FAE reaction to calculate efficiency. The difference between the samples subjected to FAE and the unreacted control was used to identify bispecific antibodies and contaminants after FAE. Figures 2a-2f Results were provided.

[0236] For reaction #1, the FAE using the DEKK variant produced only about 1% homodimer, and for reaction #3, no detectable homodimer was observed. In contrast, the FAE using 405L / 409R produced approximately 3.5% homodimer for reaction #2 and approximately 4.6% homodimer for reaction #4 (see [link to FAE]). Figure 2a / Figure 2b ).

[0237] Furthermore, samples collected after FAE from reactions #1 and #3 showed peaks (at approximately 21 min and 16 min, respectively), which were not present in individual samples and accounted for approximately 88% and 96% of the material corresponding to the DEKK heterodimer eluted from CIEX, respectively. Following FAE, earlier elution peaks (at approximately 8.5 min and 1–6 min, respectively) appeared in samples obtained from FAE reactions #5 and #9, indicating the formation of the DE half-mer. Samples collected after FAE from reactions #2 and #4 showed peaks representing the heterodimer (at approximately 16 min), accounting for approximately 83% and 81% of the material, respectively, which were not present in individual samples of the material corresponding to the 405L / 409R heterodimer eluted from CIEX.

[0238] Example 5: HP-SEC Analysis

[0239] To detect the aggregates and concentrations of IgG dimers and half-antibodies in samples before and after FAE under natural conditions, samples generated according to Example 2 (i.e., reaction products and unreacted controls) were analyzed using HP-SEC (Agilent 1260 series) with TSK-gel G3000SWxl (Tosoh Bioscience-808541), TSK guard column SWXL (Tosoh Bioscience-808543), and HP-SEC buffer (200mM sodium phosphate, 50mM NaCl, pH 7.0; composed of sodium dihydrogen phosphate dihydrate (NaH2PO4,2H2O; Sigma, ref. 71500), disodium hydrogen phosphate dihydrate (Na2HPO4,2H2O; Sigma, ref. 71643), and NaCl (Sigma, ref. S3014) dissolved in Milli-Q water, filtered through a 0.45μm membrane filter).

[0240] Inject the same amount of sample (20 μg per sample, injection volume between 10 and 100 μL). Use the obtained data to estimate the purity of the sample (percentage of intact IgG dimers or half-mers). To ensure adequate sample quality, any starting material should contain more than 95% intact molecules. Otherwise, consider further purification or repeated production. Based on the results at 280 nm, analyze the retention time and relative peak area of ​​the peaks observed in the chromatogram. Results are as follows: Figures 3a to 3d As shown, the main peak indicates IgG-IgG dimers, because if they were analyzed individually, no aggregates would be found in the same sample.

[0241] Following FAE, reactions containing CH3 variants with DE and KK (i.e., #1 and #3) showed a main peak of size corresponding to IgG, with no aggregates but a small number of half-mers. Following FAE, reactions containing CH3 variants with 405L and 409R (i.e., #2 and #4) showed a main peak of size corresponding to IgG, with no aggregates. Following FAE, in samples obtained from single-product (production) samples with DE (#5 and #9) or KK (#6 and #10), CH3 variants mainly showed half-mers, while samples with CH3 variants with 405L (#8 and #12) or 409R (#7 and #11) formed homodimers.

[0242] Example 6: Scaled-up FAE Solution

[0243] In short, IgG molecules were incubated in the presence of β-mercaptoethylamine hydrochloride without shaking. The sample buffer was then exchanged (Bex) for PBS using an ÄKTA Pure 25 system equipped with a Spark ALIAS autosampler and a single HiPrep 26 / 10 desalting column (Cytiva). For re-oxidation, the sample was held at 4°C for a sufficient time to allow for complete re-oxidation. The IgG molecules produced in Example 1 were subjected to a FAE reaction, as listed in Table 3.

[0244] Table 3. Expressed IgG antibodies used in FAE

[0245] A fresh MEA stock solution (750 mM) was prepared by dissolving 852 mg cysteine ​​hydrochloride (Sigma product catalog number #30078) in 5–6 mL PBS (pH 7.4). The pH was adjusted to 7.3–7.5 by adding NaOH (5 M) to the solution at room temperature, and the solution was filled to a final volume of 10 mL with PBS pH 7.4 to obtain the 750 mM stock solution, which was then filtered through a 0.2 μm filter before use.

[0246] Prepare the IgG mixture listed in Table 3 to a final concentration of 1.1 mg / mL by adding PBS (using Fresenius Versylene sterile endotoxin-free water, catalog number #B230531, diluted from 10× stock solution Gibco, catalog number #70011-051, pH 7.4) and adjusting to a final volume of 3.15 mL. Take 100 μL of this mixture and hold it at 4°C as a non-reactive control, i.e., without exposure to MEA or Bex, and store it at 4°C protected from light until further use. To initiate FAE, add 350 μL of 75 mM MEA to the remaining 3.05 mL, gently mix the solution, and incubate at 31°C for 5 hours without shaking.

[0247] Buffer exchange: Five hours later, using an ÄKTA Pure 25 system equipped with a Spark ALIAS autosampler and a HiPrep 26 / 10 desalting column (Cytiva catalog number #17-5087-01), sample buffer exchange (Bex) was performed to PBS (diluted to pH 7.4 using Fresenius Versylene sterile endotoxin-free water, catalog number #B230531, from 10× stock solution Gibco, catalog number #70011-051) at a flow rate of 6 mL / min and at 20°C. For reoxidation, the samples were incubated at 4°C for 64 hours (without further pipetting, shaking, or mixing) to allow complete reoxidation of the material. For quality control purposes, 60–100 μg of each sample (i.e., GF pre-control) was retained prior to gel filtration purification.

[0248] Gel filtration

[0249] The buffer-exchanged sample was concentrated to a volume of 2.4 ± 0.4 mL (~1.5 ± 0.25 mg / mL protein concentration) using an Amicon 15 Ultra (30 kDa molecular weight cutoff) device (Merck / Millipore catalog number #UFC903096). The material was then loaded onto an ÄKTA Pure 25 system equipped with a Spark ALIAS autosampler and purified by gel filtration using PBS as the mobile phase (PBS prepared above). The sample was then separated based on size using a Superdex 200 increase 16 / 40 column (Cytiva, catalog number #29321905) at a flow rate of 1 mL / min (fraction size 0.5 mL, autosampler loop 10 mL, temperature 20 °C). The sample was detected by UV light according to the manufacturer's instructions. The results for reactions #1-8 in Table 3 are as follows. Figures 4A-4H As shown in the image above.

[0250] LabChip Analysis

[0251] Under non-reducing conditions, fractions from gel filtration columns were analyzed on a LabChip GXII Touch instrument to visualize the possible presence of IgG dimers and hemimers in the obtained gel filtration fractions. LabChip analysis was performed using the HT Protein Expression Chip (Perkin-Elmer catalog number #760499) and the Protein Clear HR kit (Perkin Elmer CL8960014). LabChip analysis conditions: Input samples and fractions with concentrations greater than 1 mg / mL were diluted to 1 mg / mL using PBS. 1 μL of sample was mixed with 7 μL of non-reducing sample buffer (containing 9 mM N-ethylmaleimide) in a PCR plate and incubated at 70 °C for 10 min. Subsequently, 14 μL of H2O was added, and the plate was centrifuged at 2800 × g for 2 min. For sample analysis, the standard “HT Protein Express” script was used with the LabChip GXII Touch instrument according to the manufacturer's instructions. The results of reactions #1-8 in Table 3 are as follows. Figures 4A-4H As shown in the figure below, the samples collected after FAE mainly contained IgG dimers. Almost no aggregates were observed. The asterisks on samples #1, #2, #5, and #6 indicate IgG half-body peaks that were separated from IgG dimers during gel filtration and Labchip analysis.

[0252] Fractions containing IgG dimers but not half-mers were combined, and the protein concentration of the combined samples was determined on a Little Lunatic UV / Vis spectrophotometer according to the manufacturer's instructions (Unchained Labs). The absorbance at 280 nm was measured using a total protein program with water as a blank and an extinction coefficient of 1.45 mL / mg / cm, according to the manufacturer's instructions.

[0253] The combined samples from reactions #1-8 in Table 3 were reanalyzed using Labchip under non-reducing conditions as described above and under reducing conditions using sample buffer containing 35 mM DL-dithiothreitol (Sigma catalog number #43819). Figure 5 As shown (non-reduction: top version, reduction: bottom version). Results from the non-reduction conditions indicate that all IgG antibodies are at least 98% pure, and very small amounts of IgG with a single LC were observed. Therefore, using these samples, FAE does not cause significant loss of light chains. Results from the reduction conditions indicate that the bispecific antibodies from reactions #1-4 and #5-8 share the same mixture of light chains, suggesting the presence of two distinct Fab arms in the IgG samples.

[0254] Furthermore, following the procedures described above, the “GF pre-control samples” of reactions #1–8 collected before and after FAE were analyzed by reducing and non-reducing LabChip analysis. For all reactions, samples were confirmed to contain half-body before FAE, while samples showed fewer half-body after FAE (data not included).

[0255] Example 7: HP-CIEX Analysis

[0256] To confirm that the FAE reaction indeed resulted in the production of bispecific antibodies, all samples generated in Example 2 (reaction products and unreacted controls) were analyzed using CIEX (Agilent 1260 series). The column (TSK gel SP-STAT 7μm, 4.6mm ID×10cm L; Tosoh catalog number #21964) was equilibrated with a low-ionic-strength phosphate buffer (Buffer A) (25mM sodium phosphate, pH 6.0±0.05, composed of sodium dihydrogen phosphate dihydrate (NaH2PO4,2H2O; Sigma, ref. 71500) and disodium hydrogen phosphate dihydrate (Na2HPO4,2H2O; Sigma, ref. 71643) dissolved in Milli-Q water, filtered through a 0.45μm membrane filter). Then, the antibody was displaced from the column by increasing the Na+ concentration by running a gradient of phosphate buffer (buffer B) with gradually increasing salt percentages (25 mM sodium phosphate, 1 M NaCl, pH 6.0, composed of sodium dihydrogen phosphate dihydrate (NaH2PO4, 2H2O; Sigma, ref. 71500) and disodium hydrogen phosphate dihydrate (Na2HPO4, 2H2O; Sigma, ref. 71643) and NaCl (Sigma, ref. S3014) dissolved in Milli-Q water, filtered through a 0.45 μm membrane filter). All test and control samples were injected at a mass of 10 μg in volumes ranging from 10 to 100 μL. Based on the results at 220 nm, the peak shape, retention time, and peak area of ​​the observed main peak in the chromatogram were analyzed.

[0257] Figures 6a to 6d The results in the various lower figures show that using the DE / KK variant in a FAE results in only about 1% homodimer production (see reactions #1 and #5) or even undetectable amounts of homodimer (see reactions #2 and #6). Using the 405L / 409R variant in a FAE produces about 5–13% homodimer (see reactions #3, #4, #7, and #8).

[0258] Figure 6a and Figure 6cThe results shown in the lower figure show the appearance of peaks that were not present before FAE, which account for about 87-89% or 95-96% of the material corresponding to the DEKK heterodimer eluted from CIEX (i.e., peaks at about 21 min for #1 and #2, and peaks at about 17 min for reactions #5 and #6).

[0259] Figure 6b and Figure 6d The results shown in the lower figure show the appearance of peaks that were not present before FAE, which account for approximately 77-84% or 83-88% of the material eluted from CIEX corresponding to the 405L / 409R heterodimer, respectively (i.e., peaks at approximately 21 min for reactions #3 and #4, and peaks at approximately 16 min for reactions #7 and #8). Figures 6a-6d The asterisks indicate peaks detected in each of samples #1 through #8, suggesting that the appearance of these peaks is independent of the DE / KK or 405L / 409RFc form used. Based on their position relative to the major heterodimer peak, these peaks are expected to be removed directly using standard separation techniques.

[0260] In summary, the results indicate that using DE / KK or 405L / 409R substitutions allows for the production of heterodimers on a larger scale and with unequal proportions of starting materials than shown in Examples 1-5. Samples purified by gel filtration with 405L / 409R substitutions contained 5-13% undesirable homodimers, while in samples with DE / KK substitutions, contaminants were removed more successfully, with a maximum reported level of 1% homodimers. Higher purity was achieved using DEKK-based substitutions (87-96% vs 77-88%, respectively) compared to 405L / 409R substitutions.

[0261] Furthermore, the estimated total recovery of bispecific antibodies in milligrams of protein after FAE was comparable between the two replacement systems, but samples using the DE / KK replacement for FAE were reported to have higher purity.

[0262] Notably, ELISA confirmed that the purified bispecific antibodies produced by FAE still bound to their homologous antigens. No binding to other targets occurred. All antibodies prepared via FAE showed specific binding to their targets, leading to the conclusion that antibody specificity was maintained after FAE despite the reduction and re-oxidation of the Fab arm. Furthermore, regardless of the DE:KK ratio selected before FAE, the bispecific antibodies similarly bound to each antigen.

[0263] sequence

[0264] SEQ ID NO. 1

[0265] EVQLVQSGAEVKKPGASVKVSCKASGFTFTSYYIHWVRQAPGQGLEWIGWIYPENDNTKYNEKFKDRVTITADTSTSTAYLELSSLRSEDTAVYYCARDGYSRYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTKPPSREEMTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0266] SEQ ID NO. 2

[0267] QVQLVESGGVVQPGGSLRLSCAASGFTFSNAWMHWVRQAPGKGLEWVAQIKDKSQNYATYVAESVKGRFTISRADSKNSIYLQMNSLKTEDTAVYYCRYVHYAAGYGVDIWGQGTTVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTKPPSREEMTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0268] SEQ ID NO. 3

[0269] EVQLLEPGGGLVQPGGSLRLSCEASGSTFSTYAMSWVRQAPGKGLEWVSGFSGSGGFTFYADSVRGRFTISRDSSKNTLFLQMSSLRAEDTAVYYCAIPARGYNYGSFQHWGQGTLVTSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTDPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0270] SEQ ID NO. 4

[0271] EVQLVQSGAEVKKPGESLKISCKGSGYSFTSSYWIGWVRQMPGKGLEWMGIIFPDDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKPSDTAMYCVRLGGYSGYAEDFVDFWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTDPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0272] SEQ ID NO. 5

[0273] EVQLVQSGAEVKKPGASVKVSCKASGFTFTSYYIHWVRQAPGQGLEWIGWIYPENDNTKYNEKFKDRVTITADTSTSTAYLELSSLRSEDTAVYYCARDGYSRYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0274] SEQ ID NO. 6

[0275] QVQLVESGGGVVQPGGSLRLSCAASGFTFSNAWMHWVRQAPGKGLEWVAQIKDKSQNYATYVAESVKGRFTISRADSKNSIYLQMNSLKTEDTAVYYCRYVHYAAGYGVDIWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0276] SEQ ID NO. 7

[0277] EVQLLEPGGGLVQPGGSLRLSCEASGSTFSTYAMSWVRQAPGKGLEWVSGFSGSGGFTFYADSVRGRFTISRDSSKNTLFLQMSSLRAEDTAVYYCAIPARGYNYGSFQHWGQGTLVTSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0278] SEQ ID NO. 8

[0279] EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIFPDDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKPSDTAMYYCVRLGGYSGYAEDFVDFWGQGTLVTVSSASTKGPSVFPLASSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGS

[0280] SEQ ID NO. 9

[0281] DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWTSTRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0282] SEQ ID NO. 10

[0283] DIVMTQSPLSLPVTPGEPASISCRSSQPLVHSNRNTYLHWYQQKPGQAPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0284] SEQ ID NO. 11

[0285] SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDNDRPSGLPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0286] SEQ ID NO. 12

[0287] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

Claims

1. A method for generating a heterodimeric protein, said heterodimeric protein comprising two distinct IgG CH3 domains capable of forming a CH3-CH3 interface, said method Includes the following steps: supply: (a) A first protein comprising a first CH3 domain, wherein the first CH3 domain contains positively charged amino acid residues at positions 351 and 366, and (b) A second protein containing a second CH3 domain, wherein the second CH3 domain contains negatively charged amino acid residues at positions 351 and 368. The numbering is based on the EU numbering. - Incubate the proteins (a) and (b) together under reducing conditions to provide a first protein and a second protein that have been reduced; as well as - The reduced protein is then re-oxidized to obtain the heterodimeric protein.

2. The method according to claim 1, wherein the first protein and / or the second protein comprises or is selected from: monomeric proteins, homodimeric proteins, and heterodimeric proteins.

3. The method according to any one of the preceding claims, wherein the IgG CH3 domain is an IgG1 CH3 domain, an IgG2 CH3 domain, an IgG3 CH3 domain or an IgG4 CH3 domain, optionally wherein the CH3 domain is a human CH3 domain.

4. The method according to any one of the preceding claims, wherein the first protein and / or the second protein comprises or is selected from: antibodies and haptens, or fragments thereof, optionally wherein the antibody, hapten, or fragment thereof is a human antibody, hapten, or fragment thereof.

5. The method according to claim 4, wherein the fragment is a monomeric Fc region or a dimer Fc region.

6. The method according to claim 4 or 5, wherein the antibody, hapten, or fragment thereof is an IgG1, IgG2, IgG3, or IgG4 antibody, hapten, or fragment thereof.

7. The method according to any one of the preceding claims, wherein the first protein is an antibody having a first binding specificity, and the second protein is an antibody having a second different binding specificity.

8. The method according to any one of the preceding claims, wherein the first protein and / or the second protein is a homodimeric antibody.

9. The method according to any one of the preceding claims, wherein the first protein and / or the second protein is a heterodimeric antibody.

10. The method according to any one of the preceding claims, wherein the obtained heterodimeric protein is a heterodimeric antibody.

11. The method according to claim 9 or 10, wherein the heterodimeric antibody is a multivalent antibody, optionally wherein the multivalent antibody is a divalent, trivalent, tetravalent antibody or an antibody having up to a hexavalent antibody.

12. The method according to any one of claims 9 to 11, wherein the heterodimeric antibody is a multispecific antibody, optionally wherein the multispecific antibody is a bispecific, triple specific, or quadruple specific antibody.

13. The method according to any one of claims 9 to 12, wherein the heterodimeric antibody comprises two distinct light chains.

14. The method according to any one of the preceding claims, wherein the first CH3 domain comprises: 351K and 366R, 351R and 366K, 351K and 366K, or 351R and 366R.

15. The method according to any one of the preceding claims, wherein the second CH3 domain comprises: 351D and 368E, 351E and 368D, 351D and 368D, or 351E and 368E.

16. The method according to any one of the preceding claims, wherein the first CH3 structural domain comprises 351K and 366K, and the second CH3 structural domain comprises 351D and 368E.

17. The method according to any one of the preceding claims, wherein the first protein and / or the second protein in a) and b) are Fc-engineered proteins.

18. The method according to any one of the preceding claims, wherein the first protein and / or the second protein in a) and b) are Fc-silenced or Fc-enhanced proteins.

19. The method according to any one of the preceding claims, wherein the first protein and / or the second protein of a) and b) comprises a CH2 domain having a mutation affecting ADCC at positions 235 and / or 236.

20. The method according to any one of the preceding claims, wherein the second protein is obtained independently of the first protein.

21. The method according to any one of the preceding claims, wherein the reduction conditions include: (a) Incubate the protein in the presence of a reducing agent, optionally comprising or selected from: 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, β-mercaptoethanol, thioglycolate, cysteamine, homocysteine, penicillamine, and sodium borohydride; and / or (b) Incubate the protein at a pH between 6.0 and 12.0; and / or (c) Incubate the protein at a redox potential between -150 and -600 mV, optionally wherein the redox potential is between -250 and -400 mV.

22. The method according to any one of the preceding claims, wherein the method further comprises the steps of enriching and / or separating the heterodimeric protein obtained after re-oxidation.

23. The method of claim 22, wherein the heterodimeric protein is enriched and / or separated using methods selected from: precipitation, centrifugation, filtration, size exclusion chromatography, affinity chromatography, cation and / or anion exchange chromatography, and hydrophobic interaction chromatography.

24. An isolated heterodimeric protein, which can be obtained by any of the preceding claims.

25. An isolated heterodimeric antibody comprising a first IgG CH3 domain and a second IgG CH3 domain, wherein the first CH3 domain and the second CH3 domain are capable of forming a CH3-CH3 interface. The first CH3 domain comprises one or more of the amino acid variants 366K, 366R, 351K, or 351R, and The second CH3 domain contains one or more of amino acid variants 351E, 351D, 368E, or 368D. The heterodimeric antibody also contains two light chains with different sequences.

26. The isolated heterodimeric antibody according to claim 25, wherein the first CH3 domain comprises 351K and 366K, and the second CH3 domain comprises 351D and 368E.

27. A pharmaceutical composition comprising the isolated heterodimeric protein of claim 24, or the isolated heterodimeric antibody of claim 25 or 26, and a pharmaceutically acceptable carrier.

28. The method according to any one of claims 1-23, wherein the first protein and / or the second protein comprises an antibody-binding domain, scFv, a ligand, a protein receptor, or a cytokine.

29. The method of claim 28, wherein the heterodimeric protein is a bifunctional or multifunctional fusion protein.

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