Methods of producing dual specific proteins

Abstract

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

Technical Field

[0001] Provided herein is a method for producing a heterodimeric protein (heterodimeric protein, heterologous multimeric protein) comprising two distinct IgG CH3 domains capable of forming a CH3-CH3 interface. Furthermore, provided herein is an isolated heterodimeric protein obtained by the method. Also provided herein is 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 a multispecific binding domain; and a pharmaceutical composition comprising the isolated heterodimeric protein having multiple binding specificities. Background Art

[0002] Monospecific antibodies play an important role as therapeutic molecules for a variety of diseases, especially for the treatment of cancer. Monospecific antibodies bind to a single specific region or epitope of an antigen and are typically selected for their desired functional properties (e.g., killing tumor cells, blocking receptor-ligand interactions, or neutralizing viruses) for use in therapy. Monospecific antibodies have a variety of beneficial properties, such as they can be produced in large quantities, and their biophysical and biochemical properties can be analyzed in great detail to ensure consistency between batches, which helps with regulatory acceptability.

[0003] Despite these favorable properties, monospecific antibodies have several disadvantages related to their specificity. For this reason, in recent years, bispecific and multispecific antibodies have begun to play an even more important role because they have the potential to overcome some of the limitations of monospecific antibody therapies. For example, by combining binding to target molecules that are only present on tumor cells, they can be used as mediators to target drugs or toxic compounds to target cells, as mediators to retarget effector mechanisms to disease-associated sites, or as mediators to increase specificity for tumor cells.

[0004] Although it is worth expecting, the production and test of multi-specific antibodies still have challenges.For example, the dual-specific antibody consisting of two heavy chains and two light chains based on the IgG form has been produced by several methods.For example, it is possible to produce a new cell line or to express two antibodies in a single cell by using recombinant DNA technology by fusing the cell line of two secretory antibodies.These methods have produced a variety of antibody species, because the corresponding heavy chain from every kind of antibody can form a monospecific dimer (also referred to as homodimer (homodimer) or homodimeric antibody (homodimer antibody)), which contains two identical paired heavy chains with same specificity, and a dual-specific dimer (also referred to as heterodimer or heterodimeric antibody), which contains two different paired heavy chains with different specificities.In addition, the light chain and heavy chain from each antibody can be randomly paired to form an inappropriate, non-functional combination.This problem is referred to as heavy chain and light chain mispairing.The antibody of the total common light chain can be expressed as a dual-specific antibody to solve this problem. When a common light chain is used, expression of two heavy chains and one common light chain in a single cell may result in the production of three different antibody species (i.e., two monospecific "parent" antibodies and the bispecific antibody), necessitating purification of the bispecific antibody of interest from the resulting antibody mixture.

[0005] Although a single cell can produce essentially a single antibody species by using heterodimerization technology that pairs the constant regions of dual specificities, in order to obtain this product, it is necessary to use technologies that limit the ability to combine different binding domains (e.g., common light chain binding domains with non-common light chain antibodies). Therefore, there remains a need for a multispecific format that allows the combination of different binding domains (common and non-common light chains) to produce essentially a single product that can be tested preclinically and reliably produced for clinical and commercial development. Summary of the Invention

[0006] The present disclosure is based on a new method developed by the present inventors for producing heterodimeric proteins, in particular heterodimeric antibodies, which uses a novel Fab-arm exchange approach.

[0007] The method described herein is based on the novel Fab-arm exchange method developed by the inventors. The method involves introducing a variation (change) in the CH3 domain that allows Fab-arm exchange to occur in all IgG subtypes without destabilizing the core hinge region. As discussed in more detail in the Examples section below, the inventors have shown that when one of the CH3 domains comprises amino acids 351D and 368E and another CH3 domain comprises amino acids 366K and 351K, Fab-arm exchange can occur in IgG1 molecules. 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 on one CH3 domain, and corresponding negatively charged amino acid residues at positions 351 and 368 are present on another CH3 domain.

[0008] One advantage of the present disclosure is that it allows the Fab domain to be exchanged with any previously disclosed antibody given its amino acid sequence 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. Currently, more than 800 antibodies are known from the list of International Nonproprietary Names (INN) maintained by the World Health Organization, which are either approved or under development, and they can all be combined with another antibody as long as both antibodies contain two different IgG CH3 domains capable of forming a CH3-CH3 interface, wherein one of the antibodies has a CH3 domain with positively charged amino acids at positions 351 and 366, and the other antibody has a CH3 domain with negatively charged amino acids at positions 351 and 368.

[0009] By producing a multispecific antibody comprising a binding domain that does not have the same light chain, light chain mispairing will be produced from a single cell expression of this product, resulting in a variety of different antibody species that lose or reduce affinity or specificity, which requires laborious, time-consuming and inefficient separation methods to select the desired species. The present disclosure now allows antibodies with a common light chain binding domain to be combined with antibodies with non-common light chain binding domains, thereby producing multispecific antibody species with relative purity, yield and efficiency. In addition, the present disclosure now allows antibodies with non-common light chain binding domains to be combined with other antibodies with non-common light chains. The light chain can be any member from κ and λ families. The Fab domain can be from any source, including from a common light chain repertoire.

[0010] In addition to allowing Fab-arm exchange to be used for all IgG formats, the present disclosure also has certain unexpected advantages. For example, it has been found that, upon exposure to reducing and reoxidizing conditions, half-antibodies comprising the amino acid combinations 366K and 351K or 351D and 368E primarily remain as half-antibodies, rather than binding to other half-antibodies with the same residues. As will be apparent to those skilled in the art, this is advantageous in the context of producing bispecific antibodies, as the tendency of half-antibodies to form homodimers can hinder the efficient production and purification of bispecific antibodies. Furthermore, the presence of homodimers hinders the screening of large numbers of multispecific antibodies for functional activity. As shown in more detail in the Examples section of this application, the novel method described herein may be much better at reducing the production of potentially undesirable homodimers than the known Fab-arm exchange methods that rely on the use of the 405L / 409R variant. Specifically, as described in Example 7 of the present disclosure, the method of the present invention results in the production of only approximately 1% homodimers, compared to 5-13% obtained using the 405L / 409R method. Likewise, as also shown in Example 7, the method of the present invention can produce a larger amount of heterodimers.

[0011] Thus, in a first aspect, provided herein is a method for producing a heterodimeric protein comprising two different IgG CH3 domains capable of forming a CH3-CH3 interface, the method comprising the steps of:

[0012] -supply:

[0013] (a) a first protein comprising a first CH3 domain comprising positively charged amino acid residues at positions 351 and 366, and

[0014] (b) a second protein comprising a second CH3 domain comprising negatively charged amino acid residues at positions 351 and 368,

[0015] The numbering is based on EU numbering.

[0016] - incubating the proteins of (a) and (b) together under reducing conditions to provide a reduced first protein and a reduced second protein; and

[0017] - reoxidizing the reduced protein to obtain the heterodimeric protein.

[0018] In certain aspects, the first protein and / or the second protein includes or is selected from the group consisting of: a monomeric protein, a homodimeric protein, and a heterodimeric protein.

[0019] In certain aspects, the IgG CH3 domain is an IgG1, IgG2, IgG3, or IgG4 CH3 domain.

[0020] In certain aspects, the IgG CH3 domain is an IgG1 CH3 domain.

[0021] In certain aspects, the IgG CH3 domain of the first protein and the IgG CH3 domain of the second protein are IgG1.

[0022] In certain aspects, the IgG CH3 domain of the first protein and the IgG CH3 domain of the second protein are IgG2.

[0023] In certain aspects, the IgG CH3 domain of the first protein and the IgG CH3 domain of the second protein are IgG3.

[0024] In certain aspects, the IgG CH3 domain of the first protein and the IgG CH3 domain of the second protein are IgG4.

[0025] In certain aspects, the IgG CH3 domain is a human IgG CH3.

[0026] In some aspects, the first protein and the second protein comprise the same hinge region. In some aspects, the first protein and the second protein comprise an IgG1 hinge region. In some aspects, the first protein and the second protein comprise an IgG2 hinge region. In some aspects, the first protein and the second protein comprise an IgG3 hinge region. In some aspects, the first protein and the second protein comprise an IgG4 hinge region.

[0027] In certain aspects, the first protein and / or the second protein comprises or is selected from the group consisting of: an antibody and a half antibody, or a fragment thereof.

[0028] In certain aspects, the antibody, half antibody, or fragment thereof is a human antibody, half antibody, or fragment thereof.

[0029] In certain aspects, the fragment is a monomeric Fc region or a dimeric Fc region.

[0030] In certain aspects, the antibody, half antibody or fragment thereof is an IgG1, IgG2, IgG3 or IgG4 antibody, half antibody or fragment thereof.In certain aspects, the first protein is an antibody comprising a first binding specificity and the second protein can be an antibody comprising a second, different binding specificity.

[0031] In certain aspects, the first protein is an antibody comprising one binding specificity and the second protein can be an antibody comprising another, different binding specificity.

[0032] In some aspects, the first protein is a multispecific antibody (e.g., a bispecific or trispecific antibody). In some aspects, the second protein is a multispecific antibody (e.g., a bispecific or trispecific antibody). In some aspects, both the first and second proteins are multispecific antibodies (e.g., bispecific or trispecific antibodies).

[0033] In certain aspects, the first protein and / or the second protein is a homodimeric antibody.

[0034] In certain aspects, the first protein and / or the second protein is a heterodimeric antibody comprising a common light chain.

[0035] In certain aspects, the heterodimeric protein obtained is a heterodimeric antibody.

[0036] In certain aspects, the heterodimeric antibody is multivalent, optionally wherein the multivalent antibody comprises two or more valencies, including bivalent, trivalent, or tetravalent antibodies.

[0037] The present disclosure is suitable for producing multivalent multimers known in the art, including and excluding the use of a common light chain. WO2019 / 190327, in particular Figure 1 a-1u is incorporated by reference.

[0038] In certain aspects, the heterodimeric antibody is a multispecific antibody, optionally wherein the multispecific antibody comprises two or more valencies and is bispecific, trispecific, quadrispecific, or has up to six valencies.

[0039] In certain aspects, a heterodimeric antibody may comprise two different light chains.

[0040] In certain aspects, the first CH3 domain may comprise: 351K and 366R, 351R and 366K, 351K and 366K, or 351R and 366R.

[0041] In certain aspects, the second CH3 domain may comprise: 351D and 368E, 351E and 368D, 351D and 368D, or 351E and 368E.

[0042] In certain aspects, the second CH3 domain may include 351D and 368E, and the first CH3 domain may include 366K and 351K.

[0043] In certain aspects, the second protein is obtained independently of the first protein.

[0044] In certain aspects, reducing conditions may include:

[0045] (a) incubating the protein in the presence of any suitable reducing agent known in the art, optionally wherein the reducing agent comprises or is selected from the group consisting of: 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

[0046] (b) incubating the protein at a pH between 6.0 and 12.0, optionally wherein the pH is between 7.0 and 11.0; and / or

[0047] (c) incubating the protein at a redox potential between -150 and -600 mV, optionally wherein the redox potential is between -250 and -400 mV.

[0048] In certain aspects, the method may further comprise a step of enriching and / or isolating the heterodimeric protein obtained after reoxidation.

[0049] In certain aspects, the heterodimeric protein obtained after reoxidation is enriched and / or isolated using a method comprising or selected from the group consisting of precipitation, centrifugation, filtration, size exclusion chromatography, affinity chromatography, cation and / or anion exchange chromatography, and hydrophobic interaction chromatography.

[0050] The methods described herein provide a method for producing a heterodimeric antibody comprising two different IgG CH3 domains capable of forming a CH3-CH3 interface, the method comprising the steps of:

[0051] -supply:

[0052] (a) a first IgG antibody or half-body, wherein each CH3 domain comprises positively charged amino acid residues at positions 351 and 366, and

[0053] (b) a second IgG antibody or half, wherein each CH3 domain comprises negatively charged amino acid residues at positions 351 and 368,

[0054] The numbering is based on EU numbering.

[0055] - Incubating the antibodies and / or moieties of (a) and (b) together under conditions sufficient to disulfide isomerize the cysteines in the core hinge region of the antibodies or moieties to obtain a heterodimeric protein.

[0056] In another aspect, provided herein is an isolated heterodimeric protein obtainable by the methods of the present disclosure.

[0057] In certain aspects, the heterodimeric protein obtainable by the methods of the present disclosure is an IgG antibody.

[0058] In certain aspects, the heterodimeric protein obtainable by the methods of the present disclosure is a multi-specific IgG antibody.

[0059] In some aspects, an IgG antibody comprises two light chains with different sequences. In some aspects, an IgG antibody comprises a binding domain comprising different light chain sequences.

[0060] 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, wherein the first CH3 domain comprises one or more of the amino acid variants 366K, 366R, 351K or 351R, and wherein the second CH3 domain comprises one or more of the amino acid variants 351E, 351D, 368E or 368D, the heterodimeric antibody further comprising two light chains having different sequences.

[0061] In certain aspects, the second CH3 domain comprises 351D and 368E, and the first CH3 domain comprises 366K and 351K.

[0062] In another aspect, provided herein is a pharmaceutical composition comprising the isolated heterodimeric protein of the present disclosure and a pharmaceutically acceptable carrier.

[0063] In certain aspects, heterodimeric proteins are obtained by the methods of the present disclosure.

[0064] Throughout the description and claims of this specification, the words "comprise" and "include" and their variations mean "including but not limited to", and they are not intended to (and do not) exclude other parts, additions, components, integers or steps.

[0065] Throughout the description and claims of this specification, the singular encompasses the plural unless the context requires otherwise. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity unless the context requires otherwise.

[0066] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect or example of the disclosure are to be understood to be applicable to any other aspect or example described herein unless incompatible therewith.

[0067] Various aspects of the disclosure are described in further detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Aspects of the present disclosure are further described below with reference to the accompanying drawings, in which:

[0069] 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.

[0070] Figure 2. HP-CIEX results of reactions #1-#12 in Table 2 without FAE and after FAE. Figures 2a-2f : The two figures above show the results of the reaction without FAE, and the two figures below show the results of the reaction with FAE. Figure 2a : The two figures on the left show the results of #1, and the two figures on the right show the results of #2. Figure 2b : The left and right figures show the results of #3 and #4 respectively. Figure 2c : The left and right figures show the results of #5 and #6 respectively. Figure 2d : The left and right figures show the results of #7 and #8 respectively. Figure 2e : The left and right figures show the results of #9 and #10 respectively. Figure 2f : The left and right figures show the results of #11 and #12 respectively.

[0071] Figure 3. HP-SEC results of reactions #1-#12 in Table 2 without FAE and 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 with FAE. Figure 3a : From left to right: results of #1, #5, and #6. Figure 3b : From left to right: results of #3, #9, and #10. Figure 3c : From left to right: results of #2, #7, and #8. Figure 3d : From left to right: results of #4, #11, and #12.

[0072] Figure 4. Gel filtration purification and LabChip analysis of reactions #1-8 in Table 3 after FAE.

[0073] Figure 5 LabChip analysis of gel filtration purified FAE products from reactions #1-8 in Table 3 under non-reducing conditions (top) and reducing conditions (bottom).

[0074] Figure 6. CIEX results of samples obtained before FAE and after FAE and gel filtration. Figure 6a: Results of reactions #1 and #2 in Table 3, showing the formation of IgG heterodimers before (upper panel) and after (lower panel) FAE. Figure 6b : Results of reactions #3 and #4 in Table 3, showing the formation of IgG heterodimers before (upper panel) and after (lower panel) FAE. Figure 6c : Results of reactions #5 and #6 in Table 3, showing the formation of IgG heterodimers before (upper panel) and after (lower panel) FAE. Figure 6d : Results of reactions #7 and #8 in Table 3, showing the formation of IgG heterodimers before (upper panel) and after (lower panel) FAE. DETAILED DESCRIPTION

[0075] Human immunoglobulin G (IgG) antibodies exist in four subclasses with distinct 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.

[0076] Provided herein is a method for producing a heterodimeric protein comprising two different IgG CH3 domains capable of forming a CH3-CH3 interface. In certain aspects, the method is an in vitro method.

[0077] As used herein, the term "heterodimeric protein" refers to a protein comprising two monomers, wherein the two monomers have different polypeptides that are covalently or non-covalently linked. One or each of the monomers can be paired with a light chain. When used as the first and second proteins provided by (a) and (b) of the method as described in the present disclosure, the heterodimeric protein is included in a CH3 domain comprising positively charged amino acid residues at positions 351 and 366 or negatively charged amino acid residues at positions 351 and 368. The heterodimeric protein as obtained by the method as described in the present disclosure comprises positively charged amino acid residues at positions 351 and 366 and negatively charged amino acid residues at positions 351 and 368.

[0078] Heterodimeric protein product

[0079] As will be clear to those skilled in the art, the properties of the heterodimeric protein produced by the method of the present disclosure will be determined by the starting material, which is the first and second proteins provided by (a) and (b) of the method of the present disclosure. By way of example only, if the first and second proteins are fragments of IgG antibodies (e.g., if they comprise an IgG CH3 domain or consist of an IgG CH3 domain), the heterodimeric protein obtained by the method will be a heterodimeric fragment of an IgG antibody (e.g., wherein the heterodimeric fragment will comprise two IgG CH3 domains or consist of two IgG CH3 domains). Similarly, if the first and second proteins are IgG antibodies or IgG half bodies, the heterodimeric protein produced will be an IgG antibody. In some aspects, the first and / or second protein comprises a CH3 domain, a hinge region, and a Fab arm. In principle, the present technology allows the production of heterodimers, wherein the form of the first and / or second protein can comprise any multimerization domain, including but not limited to a variable heavy chain domain, a CH1 domain, a CH2 domain, a variable light chain, etc. In certain aspects, the first protein may comprise an antibody binding domain and the second protein may comprise a cytokine, ligand, scFv, or other domain that provides therapeutic potential (eg, a bifunctional or multifunctional fusion protein).

[0080] Examples of first and second proteins are described below.

[0081] In the context of a heterodimeric protein produced by the methods of the present disclosure, the term "heterodimeric protein" refers to a protein comprising two monomers having different polypeptides covalently or non-covalently linked, wherein the two monomers comprise two differentIgG CH3 domains or composed of them. IgG CH3 domains differ due to their different polypeptide sequences. Specifically, the two different IgG CH3 domains differ in amino acids at least at positions 351, 366, and / or 368. In certain aspects, 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. Amino acid residue numbering is based on EU numbering (available from https: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html, last updated on January 20, 2020, 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." Likewise, a CH3 domain comprising negatively charged amino acid residues at positions 351 and 368 is referred to herein as a "351 / 368 negatively charged CH3 domain" or a "second CH3 domain."

[0082] In the present disclosure, when referring to a 351 / 366 positively charged CH3 domain or a 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 the heterodimeric proteins produced by the methods described in the present disclosure are heterodimers, at least because they have two different CH3 domains.

[0083] As used herein, the term "CH3 domain" refers to an immunoglobulin, specifically the CH3 domain of an IgG immunoglobulin. The CH3 domain and its sequence are well known in the art. An "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 immunoglobulin gamma genes generally recognized in the art. In humans, the IgG immunoglobulin class includes subclasses IgG1, IgG2, IgG3, and IgG4. Typically, conventional IgG immunoglobulins are heterotetramers having two heavy chains and two light chains held together by a disulfide bond (-SS-) in the hinge region. However, in the art, IgG immunoglobulins are typically referred to as dimers (e.g., homodimers or heterodimers). The dimer is formed by two monomers, each of which comprises a heavy chain and a light chain. The heavy chain and the light chain are held together by a disulfide bond (-SS-). This monomer is referred to as a "half antibody" or "halfbody."

[0084] Because different (i.e., 351 / 366 positive charges and 351 / 368 negative charges) IgG CH3 domains can form a CH3-CH3 interface, the methods disclosed herein produce heterodimeric proteins. In certain aspects, two different IgG CH3 domains preferentially bind to each other, i.e., a tendency to bind to each other is higher than a tendency to bind to another IgG CH3 domain with the same charge. In other words, the 351 / 368 negatively charged IgG CH3 domain described herein may have a higher tendency to bind to the 351 / 366 positively charged IgG CH3 domain described herein than to another 351 / 368 negatively charged IgG CH3 domain. Similarly, the 351 / 366 positively charged IgG CH3 domain described herein may have a higher tendency to bind to the 351 / 368 negatively charged IgG CH3 domain described herein than to another 351 / 366 positively charged IgG CH3 domain.

[0085] As used herein, the term "CH3-CH3 interface" refers to the binding between two different CH3 domains that results from an interaction of amino acid residues, i.e., at least one interaction between amino acids of a first CH3 domain and amino acids of a 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 different CH3 domains form a CH3-CH3 interface, they form a heterodimeric protein (forming a heterodimer due to the different sequences of at least the CH3 domains).

[0086] 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, the CH3 domain guides the binding of antibody heavy chains, and it is known that the interface between the CH3 domains contains more than 20 contact residues from each chain that play a role in CH3-CH3 interactions (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 the present disclosure (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 dual-specific or monospecific full-length antibodies. The specificity of an antibody, defined by the VH / VL combination, generally does not affect the heavy chain dimerization behavior driven by the CH3 domain.

[0087] In certain aspects, the IgG CH3 domain of the heterodimeric protein produced by the methods of the present disclosure does not contain arginine at position 409 and / or does not contain leucine at position 405. In some examples, the IgG CH3 domain of the heterodimeric protein produced by the methods of the present disclosure contains lysine at position 409 and / or phenylalanine at position 405 (EU numbering).

[0088] 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 comprising at least two different IgG CH3 domains. In certain aspects, the fragment may also comprise one or more domains (such as CH2, CH1, VH, CL and / or VL) and / or specific binding portions that are typically present with an IgG antibody.

[0089] In some aspects, the heterodimer IgG antibody or the heterodimer fragment of IgG antibody can comprise two IgG CH3 domains, and said domains comprise or are selected from IgG1, IgG2, IgG3 and IgG4 CH3 domains. In some aspects, each IgG CH3 domain in the heterodimer IgG antibody or its heterodimer fragment is an IgG1 CH3 domain. It will be understood that each monomer forming the heterodimer IgG antibody or the heterodimer fragment of IgG antibody does not need to comprise the IgG CH3 domain belonging to the same subclass. In some aspects, each CH3 domain of the heterodimer IgG antibody or the heterodimer fragment of IgG antibody belongs to different subclasses. Only as an example, one of monomers can comprise IgG1 CH3 domain, and another can comprise IgG2, IgG3 or IgG4 CH3 domain.

[0090] In certain aspects, 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 certain aspects, the IgG CH3 domain in the heterodimeric IgG antibody or 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).

[0091] In one embodiment, the heterodimeric protein produced by the methods described herein is a heterodimeric IgG antibody. In this embodiment, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some aspects, it is a human IgG1. In some aspects, it is a human IgG2. In some aspects, it is a human IgG3. In some aspects, it is a human IgG4 antibody.

[0092] In another embodiment, the heterodimeric protein produced by the methods described herein is a heterodimeric fragment of an IgG antibody. In this embodiment, the heterodimeric fragment is a heterodimeric fragment of an IgG1, IgG2, IgG3, or IgG4 antibody. In certain aspects, it is a heterodimeric fragment of a human IgG1, IgG2, IgG3, and IgG4 antibody.

[0093] In certain aspects, when the heterodimeric protein produced by the methods described herein is a heterodimeric fragment of an IgG antibody, the fragment can comprise two IgG CH3 domains and two IgG CH2 domains (e.g., each monomer within the heterodimeric protein can comprise an IgG CH3 and an IgG CH2 domain).

[0094] In a specific embodiment, the heterodimeric protein produced by the methods described herein can comprise or consist of an IgG Fc region. In this specific embodiment, the IgG Fc region is an IgG Fc region of an IgG1, IgG2, IgG3, or IgG4 antibody. In certain aspects, it is an IgG Fc region of a human IgG1, IgG2, IgG3, or IgG4 antibody.

[0095] As used herein, the term "IgG Fc region" refers to the fragment crystallizable C-terminal region of the immunoglobulin heavy chain. The human IgG heavy chain Fc region is generally defined as comprising the amino acid residues from P230 to the carboxyl terminus of the IgG antibody. The numbering of the residues in the Fc region is the numbering of the EU index. In some aspects, the Fc region may include a hinge region. The hinge region (e.g., for IgG1, residues 216-230 according to EU numbering) may extend from the N-terminus of the Fc region. Two monomeric IgG Fc domains are held together by a disulfide bond (-SS-) in the hinge region, thereby contributing to the formation and / or maintenance of heterodimeric proteins. The number of hinge disulfide bonds varies between immunoglobulin subclasses (Papadea and Check 1989). In vivo, Fc region dimers or heterodimers interact with specific receptors on the complement system and various cell surfaces. In some aspects, the IgG Fc region may include an IgG1 or IgG2 core hinge region CPPC. Alternatively, the IgG Fc region may include an IgG3 core hinge region CPRC. Alternatively, the IgG Fc region may comprise an IgG4 core hinge region CPSC.The term "core hinge region" as used herein refers to the four amino acids corresponding to positions 226-229 (EU numbering) of a human IgG1 antibody.

[0096] In some aspects, the first protein and the second protein comprise the same hinge region. In some aspects, the first protein and the second protein comprise an IgG1 hinge region. In some aspects, the first protein and the second protein comprise an IgG2 hinge region. In some aspects, the first protein and the second protein comprise an IgG3 hinge region. In some aspects, the first protein and the second protein comprise an IgG4 hinge region.

[0097] In some aspects, the heterodimeric protein produced by the methods described herein is a heterodimeric antibody. The antibodies produced by the methods described herein can have sequences from any source, including mouse and human sequences. Antibodies can be composed of sequences from only one source, such as fully human antibodies, or they can have sequences from more than one source, thereby producing, for example, chimeric or humanized antibodies. It is desirable that the antibodies used for therapeutic purposes are as close as possible to the natural antibodies of the subject to be treated (e.g., human antibodies for human subjects).

[0098] Antibody binding can be expressed in terms of specificity and affinity. Specificity determines which antigen or epitope is bound by the binding domain. Affinity is a measure of the strength of binding to a specific antigen or epitope.

[0099] As used herein, the term "antigen" refers to a substance or molecule that triggers the immune system to produce antibodies when introduced into the body. Among them, antigens are derived from pathogenic organisms, tumor cells or other abnormal cells, from haptens or even self-structures. At the molecular level, an antigen is characterized by its ability to be bound by the antigen binding site of an antibody. A mixture of antigens can also be considered an "antigen", that is, the technician will understand that sometimes tumor cell lysates or virus particles are represented as "antigens", and such tumor cell lysates or virus particle preparations have multiple antigenic determinants. An antigen contains at least one, but usually more, epitopes. As used herein, the term "epitope" refers to a part of an antigen that is recognized by the immune system, specifically antibodies, B cells or T cells. Although epitopes are generally considered to be derived from non-self proteins, sequences that can be recognized from the host are also classified as epitopes.

[0100] It will be understood that the heterodimeric proteins (e.g., heterodimeric IgG antibodies or heterodimeric fragments thereof) produced by the methods described herein will be composed of two monomers, wherein each monomer comprises or consists of different IgG CH3 domains (i.e., 351 / 366 positively charged IgG CH3 domains and 351 / 368 negatively charged IgG CH3 domains). In addition, other differences (i.e., sequence differences) may also exist in one or both monomers (e.g., half or fragments thereof).

[0101] In some aspects, these differences can produce multivalent and / or multispecific heterodimeric proteins (such as IgG antibodies or heterodimeric fragments thereof). The term "multivalent" (e.g., a multivalent antibody or heterodimeric fragment thereof) refers to a single molecule with more than one valence, where "valence" is described as the number of antigen binding moieties present per molecule (e.g., an antibody or 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 antibodies, trivalent antibodies, tetravalent antibodies, pentavalent antibodies, etc., but at least include antibodies with hexavalence.

[0102] As used herein, the term "multi-specificity" (e.g., a multi-specific antibody or a heterodimer 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 "multi-specific antibody" includes but is not limited to a bispecific antibody, a triple-specific antibody, a quadruple-specific antibody, etc. In some aspects, the term "multi-specific antibody" refers to a bispecific antibody. In some aspects, the term "multi-specific antibody" refers to a triple-specific antibody. In some aspects, the term "multi-specific antibody" refers to an antibody with 4, 5 or 6 valences. In some aspects, the term "multi-specific antibody" refers to an antibody with more than 6 valences.

[0103] In certain aspects, the heterodimeric protein produced by the methods described herein is a heterodimeric IgG antibody, wherein the antibody is multivalent and / or multispecific.

[0104] In some aspects, heterodimeric proteins (such as IgG antibodies or heterodimeric fragments thereof) can comprise two or more variable regions. In some aspects, each of those variable regions can specifically bind to different epitopes. In some aspects, different epitopes are located on different antigens. In some aspects, different antigens are expressed on the same or different cells.

[0105] In certain aspects, the heterodimeric antibody may comprise two different light chains. Alternatively, the heterodimeric antibody may comprise two identical light chains.

[0106] In one aspect where the heterodimeric protein produced by the methods of the present disclosure is an IgG antibody, the antibody 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.

[0107] In certain aspects, the heterodimeric protein (such as IgG antibody or its heterodimeric IgG fragment) produced by the present disclosure can include two identical light chains. Alternatively, the heterodimeric protein (such as IgG antibody or its heterodimeric IgG fragment) produced by the present disclosure can include two different light chains.

[0108] In certain aspects, provided herein are methods for producing a DEKK heterodimeric protein comprising two different IgG CH3 domains capable of forming a CH3-CH3 interface, the method comprising the steps of:

[0109] -supply:

[0110] (a) a first protein comprising a first CH3 domain comprising 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, and

[0111] (b) a second protein comprising a second CH3 domain, wherein the second CH3 domain comprises 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,

[0112] The numbering is based on EU numbering.

[0113] - incubating the proteins of (a) and (b) together under reducing conditions to provide a reduced first protein and a reduced second protein; and

[0114] - reoxidizing the reduced first and second proteins to obtain the DEKK heterodimeric protein.

[0115] In certain aspects, provided herein are methods for producing a heterodimeric antibody comprising two different IgG CH3 domains capable of forming a CH3-CH3 interface, the method comprising the steps of:

[0116] -supply:

[0117] (a) a first antibody or half-body, wherein each CH3 domain comprises positively charged amino acid residues at positions 351 and 366, and

[0118] (b) a second antibody or half-body, wherein each CH3 domain comprises negatively charged amino acid residues at positions 351 and 368,

[0119] The numbering is based on EU numbering.

[0120] - incubating the antibodies and / or moieties of (a) and (b) together under reducing conditions to provide a reduced first antibody or moiety and a reduced second antibody or moiety; and

[0121] - Reoxidizing the reduced first antibody and / or second moiety to obtain the heterodimeric antibody.

[0122] In certain aspects, provided herein are methods for producing an IgG heterodimeric antibody comprising two different IgG CH3 domains capable of forming a CH3-CH3 interface, the method comprising the steps of:

[0123] -supply:

[0124] (a) a first IgG antibody or IgG half, wherein each CH3 domain comprises a positively charged amino acid residue at positions 351 and 366, and

[0125] (b) a second IgG antibody or IgG half, wherein each CH3 domain comprises a negatively charged amino acid residue at positions 351 and 368,

[0126] The numbering is based on EU numbering.

[0127] - incubating the IgG antibodies and / or IgG halves of (a) and (b) together under reducing conditions to provide a reduced first IgG antibody or IgG half and a reduced second IgG antibody or IgG half; and

[0128] - Reoxidizing the reduced IgG antibodies and / or IgG half-bodies to obtain heterodimeric IgG antibodies.

[0129] In certain aspects, provided herein are methods for producing a DEKK heterodimeric antibody comprising two different IgG CH3 domains capable of forming a CH3-CH3 interface, the method comprising the steps of:

[0130] -supply:

[0131] (a) a first antibody or half-body, wherein each CH3 domain comprises a positively charged amino acid residue at positions 351 and 366, wherein the positively charged amino acid residues at positions 351 and 366 are K and K, respectively, and

[0132] (b) a second antibody or half-body, wherein each CH3 domain comprises a negatively charged amino acid residue at positions 351 and 368, wherein the negatively charged amino acid residues at positions 351 and 368 are D and E, respectively,

[0133] The numbering is based on EU numbering.

[0134] - incubating the antibodies and / or moieties of (a) and (b) together under reducing conditions to provide a reduced first antibody or moiety and a reduced second antibody or moiety; and

[0135] - Reoxidizing the reduced antibody and / or half-body to obtain the DEKK heterodimeric antibody.

[0136] In certain aspects, provided herein are methods for producing a DEKK heterodimeric IgG antibody comprising two different IgG CH3 domains capable of forming a CH3-CH3 interface, the method comprising the steps of:

[0137] -supply:

[0138] (a) a first IgG antibody or IgG half, wherein each CH3 domain comprises a positively charged amino acid residue at positions 351 and 366, wherein the positively charged amino acid residues at positions 351 and 366 are K and K, respectively, and

[0139] (b) a second IgG antibody or IgG half, wherein each CH3 domain comprises a negatively charged amino acid residue at positions 351 and 368, wherein the negatively charged amino acid residues at positions 351 and 368 are D and E, respectively,

[0140] The numbering is based on EU numbering.

[0141] - incubating the IgG antibodies and / or IgG halves of (a) and (b) together under reducing conditions to provide a reduced first IgG antibody or IgG half and a reduced second IgG antibody or IgG half; and

[0142] - Reoxidizing the reduced IgG antibodies and / or IgG half-bodies to obtain DEKK heterodimeric IgG antibodies.

[0143] Method for preparing heterodimeric protein

[0144] The methods of the present disclosure include the steps of providing: (a) a first protein comprising a CH3 domain comprising positively charged amino acid residues at positions 351 and 366 (also referred to herein as a 351 / 366 positively charged CH3 domain), and (b) a second protein comprising a CH3 domain comprising negatively charged amino acid residues at positions 351 and 368 (also referred to herein as a 351 / 368 negatively charged CH3 domain), wherein numbering is according to EU numbering.

[0145] In certain aspects, the first and / or second protein can comprise or be selected from the group consisting of: a monomeric protein, a homodimeric protein, and a heterodimeric protein.

[0146] Those skilled in the art will appreciate that the terms "monomeric protein" and "monomer," used interchangeably herein, generally refer to a single, non-aggregated protein or polypeptide molecule. However, as described above, in the field of antibodies, the term monomer may also refer to a hemibody. For example, an IgG monomer is an IgG hemibody, i.e., a molecule comprising or consisting of an IgG heavy chain connected to an IgG light chain. A light chain and a heavy chain are linked by disulfide bonds in the IgG hemibody. Thus, in the context of the present disclosure, the term monomer refers to a single, non-aggregated protein or polypeptide molecule (e.g., a single IgG CH3 domain or a monomeric IgG Fc region), or a hemibody (e.g., an IgG hemibody), depending on the context.

[0147] Similarly, in the art, the term "homodimeric protein" or "homodimer" generally refers to a dimer formed by two identical polypeptides (e.g., two 351 / 368 negatively charged CH3 domains or two 351 / 366 positively charged CH3 domains) linked covalently or non-covalently. However, in the field of antibodies, the term homodimer can also refer to an antibody with two identical halves (e.g., an IgG antibody). Therefore, in the context of the present disclosure, the term homodimer refers to a dimer formed by two identical polypeptides (e.g., two identical IgG CH3 domains) linked covalently or non-covalently, or a homodimeric antibody (e.g., a homodimeric IgG antibody), depending on the context.

[0148] Thus, when the first protein is a homodimer, the methods disclosed herein include the following steps: providing: (a) a first protein (e.g., a homodimeric IgG antibody or a fragment thereof) comprising two (identical) CH3 domains, each domain comprising 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 homodimer, the methods disclosed herein include the following steps: providing: (b) a second protein (e.g., a homodimeric IgG antibody or a fragment thereof) comprising two (identical) CH3 domains, each domain comprising negatively charged amino acid residues at positions 351 and 368 (also referred to herein as 351 / 368 negatively charged CH3 domains).

[0149] 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 with the same charge (i.e., the CH3 domains in the first protein heterodimer are both 351 / 366 positively charged IgG CH3 domains, while the CH3 domains in the second protein heterodimer are both 351 / 368 negatively charged IgG CH3 domains). Therefore, when the first protein is a heterodimer, the method described herein comprises the following steps: (a) a first protein (e.g., a heterodimeric IgG antibody or fragment thereof) comprising two CH3 domains comprising positively charged amino acid residues at positions 351 and 366, respectively (also referred to herein as 351 / 366 positively charged CH3 domains). Similarly, when the second protein is a heterodimer, the method described in the present disclosure includes the steps of providing: (b) a second protein (e.g., a heterodimeric IgG antibody or a fragment thereof) comprising two CH3 domains, wherein the CH3 domains comprise negatively charged amino acid residues at positions 351 and 368, respectively (also referred to herein as 351 / 368 negatively charged CH3 domains).

[0150] In certain aspects, 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 certain aspects, the IgG CH3 domains of the two monomers forming the heterodimer are identical (i.e., the polypeptide sequence of the IgG CH3 domains is identical along their entire length).

[0151] In the field of antibodies, the term heterodimer can also refer to an antibody (such as an IgG antibody) comprising or consisting of different half bodies. Therefore, in the context of the present disclosure, the term heterodimer protein can also refer to a heterodimer antibody. This heterodimer antibody is prepared by two different half bodies, wherein the two half bodies comprise IgG CH3 domains with the same charge (i.e., 351 / 366 positive charge or 351 / 368 negative charge IgG CH3 domains). In some aspects, the IgG CH3 domains forming the two half bodies of the heterodimer antibody have identical amino acids at positions 351 and 366 or positions 351 and 368 (depending on whether the IgG CH3 domains are 351 / 366 positive charge or 351 / 368 negative charge). In some aspects, the IgG CH3 domains forming the two half bodies of the heterodimer antibody are identical (i.e., the polypeptide sequence of the IgG CH3 domains is identical along its entire length).

[0152] In certain aspects, the first and second proteins provided in (a) and (b) of the methods of the present disclosure are monomers (e.g., the first and second proteins are first and second half bodies, respectively). In certain aspects, the first protein (e.g., the first half body) can comprise 351 / 366 positively charged IgG CH3 domains, and the second protein (e.g., the second half body) can comprise 351 / 368 negatively charged IgG CH3 domains. Upon completion of the reduction and reoxidation steps of the methods of the present disclosure, the resulting heterodimeric protein (e.g., a heterodimeric antibody) will comprise the first and second proteins (e.g., the first and second half bodies).

[0153] In another example, the first protein provided in (a) of the method of the present disclosure is a monomer (e.g., the first protein is a half-body), 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., half-body) can contain 351 / 366 positively charged IgG CH3 domains, while the homodimeric protein (e.g., homodimeric antibody) can contain two 351 / 368 negatively charged IgG CH3 domains. After completing steps ii) and iii) of the method of the present disclosure, the resulting heterodimeric protein (e.g., heterodimeric antibody) will contain a monomeric protein (which is a half-body) and one of the monomers of the homodimeric protein (e.g., one of the half-bodies of the homodimeric antibody). Therefore, the resulting heterodimeric protein (e.g., heterodimeric antibody) will contain 351 / 366 positively charged IgG CH3 domains and 351 / 368 negatively charged IgG CH3 domains.

[0154] In another example, the first protein provided in (a) of the method of the present 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 half-body). The homodimeric protein (e.g., homodimeric antibody) can contain two 351 / 366 positively charged IgG CH3 domains, and the monomeric protein (e.g., half-body) can contain 351 / 368 negatively charged IgG CH3 domains. After completing steps ii) and iii) of the method of the present disclosure, the resulting heterodimeric protein (e.g., heterodimeric antibody) will contain one of the monomers of the homodimeric protein (e.g., one of the half-bodies of the homodimeric antibody) and the monomeric protein (which is a half-body). Therefore, the resulting heterodimeric protein (e.g., heterodimeric antibody) will contain 351 / 366 positively charged IgG CH3 domains and 351 / 368 negatively charged IgG CH3 domains.

[0155] In another example, the first protein provided in (a) of the method of the present disclosure is a homodimeric protein (e.g., a first homodimeric antibody), and the second protein provided in (b) can also be a homodimeric protein (e.g., a second homodimeric antibody). The first homodimeric protein (e.g., the first homodimeric antibody) can include two 351 / 366 positively charged IgG CH3 domains, and the second protein (e.g., the second homodimeric antibody) can include two 351 / 368 negatively charged IgG CH3 domains. After completing the reduction and reoxidation steps of the method of the present disclosure, the resulting heterodimeric protein (e.g., heterodimeric antibody) will include one of the monomers of the first homodimeric protein (e.g., one of the half bodies of the first homodimeric antibody) and one of the monomers of the second homodimeric protein (e.g., one of the half bodies of the second homodimeric antibody). Thus, the resulting heterodimeric protein (eg, heterodimeric antibody) will comprise 351 / 366 positively charged IgG CH3 domains and 351 / 368 negatively charged IgG CH3 domains.

[0156] In another example, the first protein provided in (a) of the method of the present 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., half-body) may comprise 351 / 366 positively charged IgG CH3 domains, while the heterodimeric protein (e.g., heterodimeric antibody) may comprise 351 / 368 negatively charged IgG CH3 domains. After completing the reduction and reoxidation steps of the method of the present disclosure, the resulting heterodimeric protein (e.g., heterodimeric antibody) will comprise a monomeric protein (which is a half-body) and one of the monomers of the heterodimeric protein (e.g., one of the half-bodies of the homodimeric antibody). Thus, the resulting heterodimeric protein (e.g., heterodimeric antibody) will comprise 351 / 366 positively charged IgG CH3 domains and 351 / 368 negatively charged IgG CH3 domains.

[0157] In another example, the first protein provided in (a) of the method of the present 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 half-body). The heterodimeric protein (e.g., heterodimeric antibody) can contain two 351 / 366 positively charged IgG CH3 domains, and the monomeric protein (e.g., half-body) can contain 351 / 368 negatively charged IgG CH3 domains. After completing the reduction and reoxidation steps of the method of the present disclosure, the resulting heterodimeric protein (e.g., heterodimeric antibody) will contain one of the monomers of the heterodimeric protein (e.g., one of the half-bodies of the heterodimeric antibody) and the monomeric protein (which is a half-body). Therefore, the resulting heterodimeric protein (e.g., heterodimeric antibody) will contain 351 / 366 positively charged IgG CH3 domains and 351 / 368 negatively charged IgG CH3 domains.

[0158] In another example, the first protein provided in (a) of the method of the present disclosure is a heterodimeric protein (e.g., a first heterodimeric antibody), and the second protein provided in (b) can also be a homodimeric protein (e.g., a second homodimeric antibody). The first heterodimeric protein (e.g., the first heterodimeric antibody) can include two 351 / 366 positively charged IgG CH3 domains, and the second protein (e.g., the second homodimeric antibody) can include two 351 / 368 negatively charged IgG CH3 domains. After completing the reduction and reoxidation steps of the method of the present disclosure, the resulting heterodimeric protein (e.g., heterodimeric antibody) will include one of the monomers of the first heterodimeric protein (e.g., one of the half bodies of the first heterodimeric antibody) and one of the monomers of the second homodimeric protein (e.g., one of the half bodies of the second homodimeric antibody). Thus, the resulting heterodimeric protein (eg, heterodimeric antibody) will comprise 351 / 366 positively charged IgG CH3 domains and 351 / 368 negatively charged CH3 domains.

[0159] In another example, the first protein provided in (a) of the method of the present disclosure is a homodimeric protein (e.g., a first homodimeric antibody), and the second protein provided in (b) can also be a heterodimeric protein (e.g., a second heterodimeric antibody). The first homodimeric protein (e.g., the first homodimeric antibody) can include two 351 / 366 positively charged IgG CH3 domains, and the second protein (e.g., the second heterodimeric antibody) can include two 351 / 368 negatively charged IgG CH3 domains. After completing the reduction and reoxidation steps of the method of the present disclosure, the resulting heterodimeric protein (e.g., heterodimeric antibody) will include one of the monomers of the first homodimeric protein (e.g., one of the half bodies of the first homodimeric antibody) and one of the monomers of the second heterodimeric protein (e.g., one of the half bodies of the second heterodimeric antibody). Thus, the resulting heterodimeric protein (eg, heterodimeric antibody) will comprise 351 / 366 positively charged IgG CH3 domains and 351 / 368 negatively charged CH3 domains.

[0160] In another example, the first protein provided in (a) of the method of the present disclosure is a heterodimeric protein (e.g., a first heterodimeric antibody), and the second protein provided in (b) can also be a heterodimeric protein (e.g., a second heterodimeric antibody). The first heterodimeric protein (e.g., the first heterodimeric antibody) can comprise two 351 / 366 positively charged IgG CH3 domains, and the second protein (e.g., the second heterodimeric antibody) can comprise two 351 / 368 negatively charged IgG CH3 domains. After completing the reduction and reoxidation steps of the method of the present disclosure, the resulting heterodimeric protein (e.g., heterodimeric antibody) will comprise one of the monomers of the first heterodimeric protein (e.g., one of the half of the first heterodimeric antibody) and one of the monomers of the second heterodimeric protein (e.g., one of the half of the second heterodimeric antibody). Thus, the resulting heterodimeric protein (eg, heterodimeric antibody) will comprise 351 / 366 positively charged IgG CH3 domains and 351 / 368 negatively charged CH3 domains.

[0161] In certain aspects, where the first and / or second protein provided in (a) of the method is a monomeric protein, it is a monomeric Fc region.

[0162] In certain aspects, where the first and / or second protein provided in (a) of the method is a dimer (heterodimer or homodimer), it is a dimeric Fc region.

[0163] In some aspects, the first protein and / or the second protein can comprise or be selected from: an antibody and a half antibody, or a fragment thereof. Antibodies, half antibodies, and fragments thereof are described elsewhere herein. In some aspects, the fragment is a monomeric or dimeric Fc region.

[0164] In some aspects, the first and / or second protein can have a sequence from any source, for example, mouse and human sequence. The first protein and / or the second protein can only be composed of a sequence from a source, such as a fully human antibody, or they can have a sequence from more than one source, thereby producing, for example, a chimeric or humanized antibody. It is desirable that the first protein and / or the second protein (such as the first and / or the second antibody) for treatment be as close as possible to the natural antibodies of the subject to be treated (for example, human antibodies for human subjects). Therefore, in some aspects, antibody, half antibody or its fragment is a human antibody, human half antibody or its fragment. It will be understood that the fragment must at least comprise IgG CH3 domains, hinge region and Fab arms. In some aspects, the Fab arms comprise variable heavy chain domains. In some aspects, the Fab arms comprise variable heavy chain domains and variable light chains. In some aspects, the Fab arms comprise variable heavy chain domains and do not comprise (i.e., lack) variable light chains.

[0165] In certain aspects, when the first and / or second protein is an antibody, it is a homodimeric antibody or a heterodimeric antibody.

[0166] In certain aspects, the first protein is an antibody with a first binding specificity and the second protein is an antibody with a second, different binding specificity.

[0167] In certain aspects, the first protein can be an antibody comprising a binding specificity and the second protein can be an antibody comprising a different binding specificity.

[0168] As described above, the production of heterodimeric proteins according to the methods described herein is facilitated by the preferential binding of the 351 / 366 positively charged CH3 domain (which comprises positively charged amino acid residues at positions 351 and 366) to the 351 / 368 negatively charged CH3 domain (which comprises negatively charged amino acid residues at positions 351 and 368).

[0169] In some aspects, 351 / 366 positive charge CH3 domains can include amino acid K or R at position 351 and amino acid K or R at position 366. For example, 351 / 366 positive charge CH3 domains can include 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 aspects, 351 / 366 positive charge CH3 domains can include amino acid K at position 351 (i.e., can include 351K) and amino acid K at position 366 (i.e., can include 366K). The first protein comprising two of the latter 351 / 366 positive charge CH3 domains is referred to herein as KKKK first protein. In some aspects, KKKK first protein is a homodimer or heterodimer. In some aspects, the KKKK first protein is a homodimeric IgG antibody or a fragment thereof or a heterodimeric IgG antibody or a fragment thereof. In some aspects, the KKKK IgG antibody or a fragment thereof is an IgG1, IgG2, IgG3 or IgG4 antibody or a fragment thereof.

[0170] In some aspects, the 351 / 368 negative charge CH3 domains can include the amino acid D or E at position 351 and the amino acid D or E at position 368. For example, the 351 / 368 negative charge CH3 domains can include the amino acid D at position 351 and the amino acid E at position 368, or the amino acid E at position 351 and the amino acid D at position 368, or the amino acid E at position 351 and the amino acid E at position 368, or the amino acid D at position 351 and the amino acid D at position 368. In some aspects, the 351 / 368 negative charge CH3 domains can include the amino acid D at position 351 (i.e., can include 351D) and the amino acid E at position 368 (i.e., can include 368E). The second protein comprising two of the latter 351 / 368 negative charge CH3 domains is referred to herein as the DEDE second protein. In some aspects, the DEDE second protein is a homodimer or a heterodimer. In certain aspects, the DEDE second protein is a homodimeric IgG antibody or a fragment thereof or a heterodimeric IgG antibody or a fragment thereof. In certain aspects, the DEDE IgG antibody or a fragment thereof is an IgG1, IgG2, IgG3 or IgG4 antibody or a fragment thereof.

[0171] In some aspects, the 351 / 366 positively charged CH3 domains can include the amino acid K at position 351 (i.e., can include 351K) and the amino acid K at position 366 (i.e., can include 366K), and the 351 / 368 negatively charged CH3 domains can include the amino acid D at position 351 (i.e., can include 351D) and the amino acid E at position 368 (i.e., can include 368E). As described above, the heterodimer with a 351 / 366 positively charged CH3 domain and a 351 / 368 negatively charged CH3 domain is referred to herein as a DEKK heterodimer. In some aspects, the DEKK heterodimer is a heterodimer IgG antibody or a fragment thereof. In some aspects, the DEKK IgG antibody or its fragment is an IgG1, IgG2, IgG3 or IgG4 antibody or its fragment.

[0172] It will be understood that the above amino acid positions are based on a human IgG CH3 domain. However, in one aspect utilizing a non-human IgG CH3 domain, the same amino acid substitutions are introduced into the corresponding amino acid residues.

[0173] In certain aspects, the first and second proteins are provided in a ratio that is favorable for producing a heterodimeric protein by the methods described herein. "Favorable" means that it increases the ratio of the heterodimeric protein produced (wherein the heterodimeric protein has 351 / 366 positively charged CH3 domains and 351 / 368 negatively charged CH3 domains) compared to when equal amounts of the first and second proteins are provided. In certain aspects, the ratio of the first protein (comprising 351 / 366 positively charged IgG CH3 domains) to the second protein (comprising 351 / 368 negatively charged IgG CH3 domains) is between 20:1 and 1:20 (w / w). Ratios exceeding these amounts can be used, but may actually result in reduced efficient use of the proteins. In certain aspects, the ratio is between 10:1 and 1:10. In certain aspects, the ratio is between 5:1 and 1:5. In certain aspects, the ratio is at least 1:1 (w / w), such as at least 1.2:1 (w / w), such as at least 1.5:1 (w / w) or at least 2:1 (w / w). In certain aspects, the ratio of the first protein (comprising 351 / 366 positively charged IgG CH3 domains) to the second protein (comprising 351 / 368 negatively charged IgG CH3 domains) is between 1:1 (w / w) and 2:1 (w / w).

[0174] In certain aspects, the ratio of the KKKK first protein (comprising 351 / 366 positively charged IgG CH3 domains) to the DEDE second protein (comprising 351 / 368 negatively charged IgG CH3 domains) is at least 1: 1 (w / w), such as at least 1.2: 1 (w / w), such as at least 1.5: 1 (w / w) or at least 2: 1 (w / w). In certain aspects, the ratio of the KKKK first protein (comprising 351 / 366 positively charged IgG CH3 domains) to the DEDE second protein (comprising 351 / 368 negatively charged IgG CH3 domains) is between 1: 1 (w / w) and 2: 1 (w / w).

[0175] In some aspects, the ratio of the KK first protein (comprising 351 / 366 positively charged IgG CH3 domains) to the DEDE second protein (comprising 351 / 368 negatively charged IgG CH3 domains) 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), such as at least 1.2: 1 (w / w), such as at least 1.5: 1 (w / w) or at least 2: 1 (w / w). In some aspects, the ratio of the KK first protein (comprising 351 / 366 positively charged IgG CH3 domains) to the DEDE second protein (comprising 351 / 368 negatively charged IgG CH3 domains) is between 1: 1 (w / w) and 2: 1 (w / w).

[0176] It will be understood that when the first and second proteins are hatched under reducing conditions and then reoxidized, the first and second proteins of any appropriate amount are used. Those skilled in the art, for example, determine appropriate concentrations using the method described in the following examples part. As non-limiting examples, each of the first and second proteins is used under the reducing conditions with at least 50 μg / ml, for example, at least 0.1mg / ml, at least 1.0mg / ml, at least 10mg / ml or at least 25mg / ml but no more than 100mg / ml. In some aspects, when each of the first and second proteins is an antibody, each of the first and second proteins is used under the reducing conditions with at least 50 μg / ml, for example, at least 0.1mg / ml or at least 1.0mg / ml. Typically, they can each use within the concentration range of 50 μg / ml to 2mg / ml, for example, at 50 μg / ml to mg / ml. Only as an example, they can each use with a concentration of about 1.1mg / ml.

[0177] It will also be understood that under the reducing conditions, other reagents may be present to assist in obtaining the process of the first protein reduced and the second protein reduced. For example, in a certain aspect, under reducing conditions, cystamine is added at a concentration in the range of, for example, 2 to 70 mM to further reduce any homodimer present in the reaction. For example, when the first and second proteins are antibodies (such as homodimer antibodies), cystamine is added at a concentration of about 2 to about 70 mM under reducing conditions. In some aspects, cystamine is added at a concentration of 2 to 32 mM to further reduce any homodimer present in the reaction. For example, when the first and second proteins are antibodies (such as homodimer antibodies), cystamine is added at a concentration of about 2 to about 32 mM under reducing conditions.

[0178] In some aspects, the first and second proteins are obtained independently of each other. As an example only, the first and second proteins are produced by different host cells. Exemplary methods for obtaining the first and second proteins are provided in the Examples section below. However, other methods will be known to those skilled in the art.

[0179] A "host cell" can be any host cell known in the art that is capable of expressing a recombinant DNA molecule and expressing a binding moiety.

[0180] In certain aspects, the first protein (eg, homodimeric antibody) and / or the second protein (eg, homodimeric antibody) is obtained under serum-free conditions (eg, by culturing host cells in FreeStyle 293 medium or FreeStyle Cho medium, Invitrogen).

[0181] In certain aspects, between providing the first and second proteins in (a) and (b) and incubating under the reducing conditions of the method, the first and / or second proteins are purified using methods known in the art. These methods may include precipitation, centrifugation, filtration, size exclusion chromatography, affinity chromatography, cation and / or anion exchange chromatography, hydrophobic interaction chromatography, and the like. For antibody mixtures comprising IgG molecules, protein A or protein G affinity chromatography can be used (see, e.g., U.S. Patents 4,801,687 and 5,151,504).

[0182] The incubation under the reducing and reoxidation conditions of the method facilitates the generation of a heterodimeric protein by recombination of the first and second proteins (e.g., antibodies or dimeric fragments thereof) or binding of the first and second proteins (e.g., half or monomeric fragments thereof). Recombination of the first and second proteins occurs when the first and second proteins exchange monomers (e.g., half or dimeric fragments thereof) to generate a heterodimeric protein comprising two different IgG CH3 domains capable of forming a CH3-CH3 interface.

[0183] As used herein, the term "incubating" refers to keeping, retaining or maintaining the first and second proteins together under relevant conditions (ie, reducing conditions). The first and second proteins are incubated together in a composition or formulation comprising the first and second proteins.

[0184] The incubating under reducing conditions of the method comprises incubating the proteins of (a) and (b) together under reducing conditions to provide a reduced first protein and / or a reduced second protein.

[0185] The term "reducing conditions" refers to an environment in which the first and / or second protein is more likely to be reduced than oxidized. In certain aspects, reducing conditions can result in "disulfide bond reduction" (i.e., a process in which a disulfide bond is broken, thereby generating two thiol groups (-SH groups)). One skilled in the art will appreciate that when an antibody (homodimer or heterodimer antibody) is incubated under reducing conditions, reduction of disulfide bonds (such as disulfide bonds in the core hinge region) can result in separation of the antibody into two halves.

[0186] The step of incubating the first and second proteins under reducing conditions can 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 changes molecules in its environment to become more reduced. The reducing agent can act by donating electrons, thereby being oxidized after reducing the substrate (i.e., the first and / or second protein).

[0187] Examples of reducing agents include 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, β-mercaptoethanol, thioglycolate, cysteamine, homocysteine, penicillamine and / or sodium borohydride.

[0188] In certain aspects, when the reducing agent is 2-MEA, its concentration is from about 25 mM to about 100 mM, such as from about 50 mM to about 75 mM. In certain aspects, the concentration of 2-MEA is about 75 mM.

[0189] It will be appreciated that the time of incubation with the reducing agent may depend on the concentration and / or temperature at which the incubation is carried out. For example, a higher concentration of reducing agent may allow for a shorter incubation time and / or a lower incubation temperature.

[0190] In certain aspects, when the concentration of 2-MEA is about 75 mM, the incubation is performed at about 31°C for at least 300 minutes. In certain aspects, when the concentration of 2-MEA is about 75 mM, the incubation is performed at about 31°C for about 300 minutes.

[0191] In one aspect, the reducing agent does not comprise an enzyme.

[0192] In some aspects, the pH is 7.4. In addition or as an alternative, hatching the first and second albumen under reducing conditions can include hatching the first and second albumen under 6.0 or higher pH, for example, at 7.0 or higher pH, at 8.0 or higher pH, at 9.0 or higher pH, at 10.0 or higher pH, at 11.0 or higher pH, or at 12.0 pH. For example, the first and second albumen are hatched at a pH between 6.0 to 11.0, or at a pH between 6.0 to 10.0, optionally wherein the pH is between 7.0 to 8.0 (for example, between pH 7.3 to 7.5). In some aspects, the pH is 7.4. In addition or as an alternative, hatching the first and second albumen under reducing conditions can include hatching albumen under the redox potential between -150 and -600mV, optionally wherein the redox potential is between -250 to -400mV. The example of applicable reducing conditions is known in the art. Some examples are described in Labrijn AF., Nature Protocols 2014, Vol. 9, No. 10, pp. 2450-2463.

[0193] The method further comprises reoxidizing the reduced protein to obtain the heterodimeric protein.

[0194] The term "reoxidation" or "oxidation" as used herein refers to undergoing or causing to undergo a reaction in which electrons are lost to another species. In the context of the present disclosure, the reoxidation step can allow the resulting reduced first protein and reduced second protein to bind together to form a heterodimeric protein comprising two different IgG CH3 domains capable of forming a CH3-CH3 interface.

[0195] Due to the preferential binding of the first protein with a 351 / 366 positively charged IgG CH3 domain and the second protein with a 351 / 368 negatively charged IgG CH3 domain, a greater proportion of heterodimeric proteins comprising two different IgG CH3 domains will be formed compared to dimeric proteins (homodimers or heterodimers) of IgG CH3 domains with the same charge. Therefore, the present disclosure provides a method for efficiently and controllably producing a mixture of well-defined Ig antibodies or heterodimeric fragments thereof with a high dual specificity ratio in the mixture. In systems where dual specificity is desired, a dual specificity ratio of at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or more can even be obtained. This means that only 5% or less, or 3% or less, of monospecific bivalent byproducts are obtained. It is noteworthy that the monomeric byproducts, i.e., half molecules, produced by the methods described herein are more stable than those produced by at least some other methods known in the art (such as the method 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 the need to repeat the oxidation step.

[0196] In some aspects, the reoxidation of the reduced protein is achieved by removing the reducing agent from the mixture of the reduced first protein and the reduced second protein. As an example only, the reducing agent is removed by diafiltration (e.g., described in Labrijn AF., Nature Protocols 2014, Vol. 9, No. 10, pp. 2450-2463). It will be understood that in order to reoxidize the reduced first and second proteins, it is not necessary to completely remove the reducing agent. For example, in the context of 2-MEA as a reducing agent, reducing the concentration to less than 50 μM can be sufficient to reoxidize the reduced first protein and the reduced second protein. Another example of a method for reoxidizing the reduced protein is buffer exchange. In some aspects, buffer exchange is for PBS. Optionally, the pH of PBS is 7.4. Methods for performing buffer exchange are known to those skilled in the art. In some aspects, buffer exchange is performed using Zeba plates, as illustrated in the Examples section of the present disclosure. As another example, a Spark ALIAS autosampler and a desalting column (e.g., HiPrep 26 / 10) can be used. PBS was buffer exchanged using a Pure 25 system. In this example, the buffer exchange occurred at a flow rate of 6 mL / min and 20 degrees Celsius.

[0197] After buffer exchange is complete, the sample can be maintained at a temperature of about 4°C for about 24, 48, 64 hours, or longer to allow for complete reoxygenation.

[0198] Alternatively or additionally, the reoxidation step comprises incubating the reduced first protein and the reduced second protein with an oxidizing agent.

[0199] In some aspects, the method also includes the step of enrichment and / or separation of the heterodimeric protein obtained. Can be by conventional methods, such as conventional purification methods, from any pollutant that may be produced by methods described herein, enrichment and / or separation of heterodimeric protein. These pollutants can include homodimeric protein (protein comprising two monomers, each monomer has identical IgG CH3 domain) and / or monomeric protein (for example half body). The method for the heterodimeric protein that purification is produced can include precipitation, centrifugal, filtration, size exclusion chromatography, affinity chromatography, cation and / or anion exchange chromatography, hydrophobic interaction chromatography etc. For the antibody mixture comprising IgG molecules, protein A or protein G affinity chromatography (see, for example, United States Patent (USP) 4,801,687 and 5,151,504) can be used.

[0200] In some aspects, the incubation under reducing conditions and reoxidation of the reduced proteins together is also described as incubating the first and second proteins under conditions sufficient to allow the cysteines in the CH3 region to undergo disulfide bond isomerization to obtain a heterodimeric protein. In some aspects, when the first and second proteins are antibodies or half antibodies, the incubation under reducing conditions and reoxidation of the reduced proteins together is described as incubating the first and second proteins under conditions sufficient to allow the cysteines in the core hinge region of the first and second proteins to undergo disulfide bond isomerization to obtain a heterodimeric protein.

[0201] In some aspects, the first protein and / or the second protein comprise a fusion protein. The fusion protein may comprise an antibody binding domain, scFv, a ligand, a protein receptor, or a cytokine. In some aspects, the first protein and / or the second protein comprise an antibody binding domain, scFv, a ligand, a protein receptor, or a cytokine. In some aspects, the heterodimer protein is a bifunctional or multifunctional fusion protein.

[0202] In another aspect, provided herein is an isolated heterodimeric protein obtainable by the methods of the present disclosure.

[0203] In certain aspects, the heterodimeric protein obtainable by the methods of the present disclosure is an IgG antibody, eg, IgG1, IgG2, IgG3, or IgG4.

[0204] In certain aspects, an IgG antibody may comprise two light chains with different sequences.

[0205] In another aspect, provided herein is an isolated heterodimeric antibody 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 the amino acid variants 351E, 351D, 368E or 368D, and the 351 / 366 positively charged CH3 domain comprises one or more of the amino acid variants 366K, 366R, 351K or 351R, the heterodimeric antibody further comprising two light chains having different sequences.

[0206] In certain aspects, the 351 / 368 negatively charged CH3 domain comprises 351D and 368E, and the 351 / 366 positively charged IgG CH3 domain comprises 366K and 351K.

[0207] It will be understood that in the context of the methods described herein, the aspects mentioned herein in relation to heterodimeric proteins also apply to the heterodimeric proteins obtainable by the methods described herein, as well as to the 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 the amino acid variants 351E, 351D, 368E or 368D, and the 351 / 366 positively charged CH3 domain comprises one or more of the amino acid variants 366K, 366R, 351K or 351R, the heterodimeric antibodies further comprising two light chains having different sequences.

[0208] Changes at positions 351 / 366 and / or 351 / 368 can be combined with any of the modifications described in WO2020 / 226502A2, which is incorporated herein by reference in its entirety. Similarly, changes at positions 351 / 366 and / or 351 / 368 can be combined with any of the modifications described in WO2021 / 235936A1, which is incorporated herein by reference in its entirety.

[0209] In another aspect, provided herein is a pharmaceutical composition comprising the isolated heterodimeric protein of the present disclosure and a pharmaceutically acceptable carrier. The term "pharmaceutical composition" refers to a preparation in which the biological activity of the active ingredient (e.g., heterodimeric protein of the present disclosure) contained is effective, and does not contain other ingredients that have unacceptable toxicity to the subject to which the preparation is administered. The term "pharmaceutically acceptable carrier" refers to any carrier useful for the dissolution of a reagent (e.g., heterodimeric protein of the present disclosure) and for delivery to a subject. Various pharmaceutically acceptable carriers are known in the art. As examples only, these include saline, phosphate buffered saline, or phosphate-buffered saline. The composition can also conventionally contain pharmaceutically acceptable concentrations of salt, buffer, preservative, compatible carrier, supplemental immunopotentiator, such as adjuvant and cytokine, and optionally other therapeutic agents. The composition can also include an antioxidant and / or preservative. As antioxidants, there may be mentioned thiol derivatives (e.g., monothioglycerol, cysteine, acetylcysteine, cystine, dithioerythritol, dithiothreitol, glutathione), tocopherol, butylated hydroxyanisole, butylated hydroxytoluene, sulfites (e.g., sodium sulfate, sodium bisulfite, sodium acetone sodium bisulfite, sodium metabisulfite, sodium sulfite, sodium formaldehyde sulfoxylate, sodium thiosulfate) and nordihydroguaiaretic acid. Suitable preservatives may be, for example, phenol, chlorobutanol, benzyl alcohol, methylparaben, propylparaben, benzalkonium chloride and cetylpyridinium chloride.

[0210] Terms

[0211] 1. A method for producing a heterodimeric protein comprising two different IgG CH3 domains capable of forming a CH3-CH3 interface, the method comprising the following steps:

[0212] -supply:

[0213] (a) a first protein comprising a first CH3 domain comprising positively charged amino acid residues at positions 351 and 366, and

[0214] (b) a second protein comprising a second CH3 domain comprising negatively charged amino acid residues at positions 351 and 368,

[0215] The numbering is based on EU numbering.

[0216] - incubating the proteins of (a) and (b) together under reducing conditions to provide a reduced first protein and a reduced second protein; and

[0217] - reoxidizing said reduced first and second proteins to obtain a heterodimeric protein.

[0218] 2. The method according to clause 1, wherein the first protein and / or the second protein comprises or is selected from the group consisting of: a monomeric protein, a homodimeric protein, and a heterodimeric protein.

[0219] 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.

[0220] 4. The method according to any one of the above clauses, wherein the first protein and / or the second protein comprises or is selected from: an antibody and a half antibody or a fragment thereof, optionally wherein the antibody, half antibody or a fragment thereof is a human antibody, half antibody or a fragment thereof.

[0221] 5. The method according to clause 4, wherein the fragment is a monomeric Fc region comprising a hinge or a dimeric Fc region comprising a hinge.

[0222] 6. The method according to clause 4 or 5, wherein the antibody, half antibody or fragment thereof is an IgG1, IgG2, IgG3 or IgG4 antibody, half antibody or fragment thereof.

[0223] 7. The method according to any of the preceding clauses, wherein the first protein is an antibody with a first binding specificity and the second protein is an antibody with a second, different binding specificity.

[0224] 8. The method according to any one of the preceding clauses, wherein the first protein and / or the second protein is a homodimeric antibody.

[0225] 9. The method according to any one of the preceding clauses, wherein the first protein and / or the second protein is a heterodimeric antibody.

[0226] 10. The method according to any one of the preceding clauses, wherein the heterodimeric protein obtained after reoxidation of the reduced protein is a heterodimeric antibody.

[0227] 11. The method according to clause 9 or 10, wherein the heterodimeric antibody is multivalent, optionally wherein the multivalent antibody is a bivalent, trivalent, tetravalent antibody or has up to six valencies.

[0228] 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 quadrispecific antibody.

[0229] 13. The method according to any one of clauses 9 to 12, wherein the heterodimeric antibody comprises two different light chains.

[0230] 14. The method according to any one of clauses 9 to 12, wherein the heterodimeric antibody comprises a single light chain.

[0231] 15. The method according to any one of clauses 9 to 12, wherein the heterodimeric antibody comprises three light chains.

[0232] 16. The method according to any one of clauses 9 to 12, wherein the heterodimeric antibody comprises three different light chains.

[0233] 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 that is different from the two identical light chains.

[0234] 18. The method according to any of the preceding clauses, wherein the light chain is any member from the kappa or lambda family.

[0235] 19. A method according to any one of the preceding clauses, wherein the first protein and the second protein comprise the same hinge region.

[0236] 20. The method according to any of the preceding clauses, wherein in certain aspects, the first protein and the second protein comprise an IgG1 hinge region.

[0237] 21. A method according to any one of the preceding clauses, wherein the first protein and the second protein comprise an IgG2 hinge region.

[0238] 22. A method according to any one of the preceding clauses, wherein the first protein and the second protein comprise an IgG3 hinge region.

[0239] 23. A method according to any one of the preceding clauses, wherein the first protein and the second protein comprise an IgG4 hinge region.

[0240] 24. The method according to any one of the preceding clauses, wherein the first CH3 domain comprises: 351K and 366R, 351R and 366K, 351K and 366K, or 351R and 366R.

[0241] 25. The method according to any of the preceding clauses, wherein the second CH3 domain comprises: 351D and 368E, 351E and 368D, 351D and 368D, or 351E and 368E.

[0242] 26. The method according to any of the preceding clauses, wherein the first CH3 domain comprises 351K and 366K, and the second CH3 domain comprises 351D and 368E.

[0243] 27. The method according to any of the preceding clauses, wherein the first and / or second protein of a) and b) comprises one or more Fc modifications.

[0244] 28. The method according to any one of the preceding clauses, wherein the first and / or second protein of a) and b) is an Fc-engineered protein.

[0245] 29. The method according to any of the preceding clauses, wherein the first and / or second protein of a) and b) is an Fc-silenced or Fc-enhanced protein.

[0246] 30. The method according to any of the preceding clauses, wherein the first and / or second protein of a) and b) comprises a CH2 domain having a mutation at position 235 and / or 236 that affects ADCC.

[0247] 31. The method according to any of the preceding clauses, wherein the second protein is obtained independently of the first protein.

[0248] 32. The method according to any one of the preceding clauses, wherein the reducing conditions comprise:

[0249] (a) incubating the protein in the presence of a reducing agent, optionally wherein the reducing agent comprises or is selected from the group consisting of: 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

[0250] (b) incubating the protein at a pH between 6.0 and 12.0, optionally wherein the pH is between 7.0 and 8.0; and / or

[0251] (c) incubating the protein at a redox potential between -150 and -600 mV, optionally wherein the redox potential is between -250 and -400 mV.

[0252] 33. The method according to any one of the preceding clauses, wherein the method further comprises a step of enriching and / or isolating the heterodimeric protein obtained after reoxidizing the reduced protein.

[0253] 34. The method according to clause 33, wherein the heterodimeric protein is enriched and / or isolated using a method comprising or selected from the group consisting of: precipitation, centrifugation, filtration, size exclusion chromatography, affinity chromatography, cation and / or anion exchange chromatography and hydrophobic interaction chromatography.

[0254] 35. An isolated heterodimeric protein obtainable by a method according to any of the preceding clauses.

[0255] 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,

[0256] wherein the first CH3 domain comprises one or more of the amino acid variants 366K, 366R, 351K or 351R, and

[0257] wherein the second CH3 domain comprises one or more of the amino acid variants 351E, 351D, 368E or 368D,

[0258] The heterodimeric antibody also comprises two or more light chains with different sequences.

[0259] 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.

[0260] 38. A pharmaceutical composition comprising the isolated heterodimeric protein according to item 35, or the isolated heterodimeric antibody according to item 36 or 37, and a pharmaceutically acceptable carrier.

[0261] 39. A method according to any one of clauses 1 to 34, wherein the first protein and / or the second protein comprises an antibody binding domain, a scFv, a ligand, a protein receptor or a cytokine.

[0262] 40. The method according to clause 39, wherein the heterodimeric protein is a bifunctional or multifunctional fusion protein.

[0263] Example

[0264] Example 1: Transfection, expression and purification of IgG antibodies for Fab arm exchange (FAE)

[0265] The purpose of the following experiments was to directly compare the results of FAE using the different variants listed in Table 1 (ie, DEKK variant vs. variant 405L-409R).

[0266] Using ExpiFectamine TM 293 transfection kit (ThermoFisher Scientific, catalog #A14635) and OptiMEM I reduced serum medium (Gibco, catalog #31985062) were added to 100 mL of Expi293F TM Expi293F cells were cultured in expression medium (ThermoFisher Scientific, catalog #A14351010). TM Cells (ThermoFisher Scientific) were transiently transfected with various expression vectors encoding heavy (HC) and light chains (LC) (see Table 1).

[0267] Table 1. Expression vectors used to produce FAE antibodies

[0268] # Expressed half antibody (HAb) CH3 modification HC sequence LC sequence 1 HAb1-KK KK SEQ ID NO: 1 SEQ ID NO: 9 2 HAb2-KK KK SEQ ID NO: 2 SEQ ID NO: 10 3 HAb3-DE DE SEQ ID NO: 3 SEQ ID NO: 11 4 HAb4-DE DE SEQ ID NO: 4 SEQ ID NO: 12 5 HAb5-F405L F405L SEQ ID NO: 5 SEQ ID NO: 9 6 HAb6-F405L F405L SEQ ID NO: 6 SEQ ID NO: 10 7 HAb7-K409R K409R SEQ ID NO: 7 SEQ ID NO: 11 8 HAb8-K409R K409R SEQ ID NO: 8 SEQ ID NO: 12

[0269] Table 1: Expression vectors (#1-8) were used for transfection and production of HC and LC molecules for final IgG production. The expression vectors contained DNA constructs encoding CH3 domains with the indicated modifications, model heavy chains (HC) and model light chains (LC).

[0270] Briefly, 3 mL of Opti-MEM TM Dilute 50 μg of plasmid DNA (0.1 mL from a 0.5 mg / mL DNA stock) with 1% Opti-MEM and 0.16 mL of ExpiFectamine TM 293 reagent, and then incubate for 5 minutes to prepare the DNA-Opti-MEM mixture and the Expifectamine-Opti-MEM mixture. Then, 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, add the full volume of ExpiFectamine TM The 293 / plasmid DNA complex (approximately 6.06 mL) was added dropwise to the cells while gently vortexing the flask during addition and then incubated (37° C. incubator, ≥80% relative humidity and 8% CO 2 , at 155 rpm on an orbital shaker).

[0271] 18-22 hours after transfection, add ExpiFectamine TMEnhancer 1 and Enhancer 2 of the 293 transfection kit were added to the transfection bottle while gently vortexing. On the 6th day after transfection, the culture medium was collected and the cells were centrifuged at 500g for 10 minutes at RT. The supernatant was collected and transferred to a new 50ml test tube and the cells were centrifuged at 3000g for 20 minutes. The supernatant was filtered using a 0.45μm filter on the top of the bottle and the IgG concentration was measured using the ForteBIO Octet-QK system based on biomembrane interferometry (BLI). This enables real-time quantitative and kinetic characterization of the interaction of biomolecules. The supernatant was used for purification.

[0272] A Protein A column (GE Healthcare / Cat. No. 11-0034-95, according to GE Healthcare instructions) was used. Pure system (Cytiva, EN490, serial number: 2031829) purifies the culture supernatant containing 9-12mg IgG and is eluted in 0.1M citrate buffer (pH 3.0), then immediately neutralized in an equal volume of 1.0M Tris-HCl (pH 8.0), or directly rebuffered to PBS using a desalting column (Cytiva #17-1408-01). As described in Example 2, the purified IgG molecules were used in FAE.

[0273] According to the Lambert-Beer law, PBS was used as a blank and the universal molar extinction coefficient of 1.45 mol was used. -1 dm 3 cm -1 All samples were adjusted for amino acid composition 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 in PBS pH 7.4.

[0274] Example 2: FAE solution

[0275] 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 exchanged into PBS pH 7.4 (Bex) using a Zeba plate at room temperature. For reoxidation, the sample was kept at 4°C for at least 1 night.

[0276] The IgG molecules produced in Example 1 were subjected to FAE reactions in a 96-well format, as listed in Table 2.

[0277] Table 2. Expressed IgG antibodies used in FAE

[0278] FAE# Half antibody CH3 Half antibody CH3 1 HAb3-DE DE HAb1-KK KK 2 HAb7-K409R K409R HAb5-F405L F405L 3 HAb4-DE DE HAb2-KK KK 4 HAb8-K409R K409R HAb6-F405L F405L 5 HAb3-DE DE 6 HAb1-KK KK 7 HAb7-K409R K409R 8 HAb5-F405L F405L 9 HAb4-DE DE 10 HAb2-KK KK 11 HAb8-K409R K409R 12 HAb6-F405L F405L

[0279] Table 2. Expressed IgG (half) antibodies with the indicated CH3 modifications. FAE reactions #1-4 included a mixture of two IgG molecules, while FAE reactions #5-12 included a single IgG as a control.

[0280] Fresh MEA stock solution (750 mM) was prepared by dissolving 852 mg of cysteamine hydrochloride (Sigma product catalog # 30078) in 5-6 mL of PBS (pH 7.4). The pH was adjusted to pH 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 the final volume was 10 mL to achieve a 750 mM stock solution. Before use, the solution was filtered through a 0.2 μm filter.

[0281] Briefly, the IgG samples listed in Table 2 were prepared in a deep well plate (plate 1, 175 μL final volume adjusted by adding PBS pH 7.4) at a concentration of 1.1 mg / mL. In 12 wells of another deep well plate (plate 2), 11 μL of the reducing agent 750 mM MEA was aspirated. 100 μL of each prepared sample in plate 1 was gently mixed with the MEA in plate 2, covered with an aluminum seal and kept at 31°C for 5 hours without shaking. The remaining material in plate 1 (75 μL) was used as an unreacted control, i.e., not exposed to MEA or Bex, and stored in the dark at 4°C until further use. After incubation, the samples were washed with water using Zeba TM Desalting plates, 96 well (ThermoFisher Scientific, catalog #89807) were spun down and 100 μL of each sample was used for buffer exchange (bex).

[0282] Buffer exchange:

[0283] Using Zeba TM The samples were buffer exchanged with PBS pH 7.4 (1X, Gibco catalog #10010-015) using a spin desalting kit (Thermo Fisher Scientific, catalog #89807) to remove the reducing agent and reform the disulfide bonds.

[0284] In short, Zeba TM The desalting plate was equilibrated to room temperature by spinning and assembled on top of the wash plate. The plate assembly was centrifuged to remove the storage solution, and the wash plate was blotted dry on a paper towel. Three wash steps were performed by adding wash buffer (1×, Gibco catalog #10010-015), and then the plate was centrifuged, the flow-through was discarded, and the plate was blotted dry on a paper towel.

[0285] Then, Zeba TM The desalting plate was stacked on top of the collection plate and the IgG sample was loaded into the wells and centrifuged. The flow-through containing IgG was transferred to another Zeba TM To achieve a more complete buffer exchange, transfer the flow-through to another Zeba TM All centrifugations were performed at 1000 × g for 2 minutes. TM The third flow-through containing the sample from the three plates was retained for IgG concentration measurement. The buffer-exchanged samples were stored in a covered collection plate and kept overnight at 4°C (without further pipetting, shaking or mixing) to allow the material to fully reoxidize. As described in Example 1, the buffer-exchanged samples and unreacted controls stored on plate 1 at 4°C were measured on a Lunatic system. Under the indicated reducing and non-reducing conditions, the obtained IgG molecules were used for SDS-PAGE using Labchip and analyzed using CIEX and HP-SEC as appropriate.

[0286] Example 3: LabChip analysis

[0287] Approximately 1 μg of purified reaction products (including a non-reacted control) were analyzed for IgG under non-reducing conditions using a Labchip (LabChip GXII Touch HT; Perkin Elmer) using the Protein Clear HR Kit (dye solution, sample buffer, protein gel matrix, protein ladder, low molecular weight marker, wash buffer; Perkin Elmer CLS960014) and the Protein Expression Assay LabChip used with the GXII Touch HT (Elmer760499) according to the manufacturer's instructions.

[0288] From samples run under non-reducing conditions, IgG, half-bodies, and IgG-1 LC bands were quantified using Labchip RX reviewer software. Only samples were analyzed in which the IgG-1 LC band accounted for less than 15% of the signal in the sample subjected to FAE. In addition, care was taken to include only samples in which the starting material contained greater than 95% intact molecules (IgG dimers or half-bodies). Figure 1 Results are provided.

[0289] Samples collected before the FAE reaction showed that species containing the CH3-DE variant primarily formed DEDE homodimers, while samples containing the CH3 KK variant primarily formed hemimers. Single-arm products (production) with CH3 variants 405L or 409R primarily formed homodimers. Samples containing mixtures of DE / KK or 405L / 409R showed the following: Figure 1Bands shown; samples from reactions containing the DE / KK CH3 variants showed a mixture of homodimers and hemimers, whereas samples from proteins containing the CH3 variants 405L / 409R showed predominantly homodimers.

[0290] The single product (production) from samples obtained after reaction of FAE with DE or KK CH3 mutations appeared mainly as hemimers, whereas samples with 405L or 409R CH3 mutations formed homodimers.

[0291] The sample with a mixture of CH3-containing DE / KK species obtained after FAE reaction showed an IgG peak with half-body contaminants, whereas an IgG peak with unresolved homodimer contaminants was detected in the sample containing 405L / 409R species.

[0292] Example 4: HP-CIEX analysis

[0293] To confirm that the FAE reaction resulted in the production of bispecific antibodies, all samples (reaction products and unreacted controls) produced in Example 2 were analyzed by CIEX (Agilent 1260 series). A chromatography column (TSK gel SP-STAT 7 μm, 4.6 mm ID × 10 cm L; Tosoh catalog #21964) was equilibrated with a low ionic strength phosphate buffer (buffer A) (25 mM sodium phosphate, pH 6.0 ± 0.05, consisting of sodium dihydrogen phosphate dihydrate (NaH2PO4, 2H2O; Sigma, ref. 71500) and sodium hydrogen phosphate dihydrate (Na2HPO4, 2H2O; Sigma, ref. 71643) dissolved in Milli-Q water, filtered through a 0.45 μm membrane filter). The antibody was then displaced from the column by increasing the Na+ concentration by running a phosphate buffer gradient with increasing percentages of saline (Buffer B) (25 mM sodium phosphate, 1 M NaCl, pH 6.0, consisting of sodium phosphate monobasic dihydrate (NaH2PO4, 2H2O; Sigma, ref. 71500) and sodium phosphate dibasic 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 samples and controls were injected with a 10 μg sample mass and injection volumes ranging from 10 to 100 μL. The chromatograms were analyzed for peak shape and retention time. The peak area of ​​the main peak was observed based on the results at 220 nm.

[0294] The percentage of dual-specific antibodies in samples subjected to FAE was used to calculate the efficiency as a percentage of the FAE reaction. The difference between samples subjected to FAE and unreacted controls was used to identify dual-specific antibodies and contaminants after FAE. Figure 2a -f provides the results.

[0295] For reaction #1, the FAE using the DEKK variant produced only about 1% homodimers and no detectable homodimers for reaction #3, whereas the FAE using 405L / 409R produced about 3.5% homodimers for reaction #2 and about 4.6% homodimers for reaction #4 (see Figure 2a / b).

[0296] In addition, samples collected from reactions 1# and #3 after FAE showed the appearance of peaks (at approximately 21 min and approximately 16 min, respectively) that were not present in the individual samples and accounted for approximately 88% or 96%, respectively, of the material corresponding to the DEKK heterodimer eluted from CIEX. In samples obtained from FAE reactions #5 and #9 after FAE, earlier eluting peaks (at approximately 8.5 min and 1-6 min, respectively) appeared, suggesting the formation of DE hemimers. Samples collected from reactions #2 and #4 after FAE showed peaks representing heterodimers (at approximately 16 minutes), accounting for approximately 83% or 81%, respectively, that were not present in the individual samples of material corresponding to the 405L / 409R heterodimer eluted from CIEX.

[0297] Example 5: HP-SEC analysis

[0298] To detect the aggregates and content of IgG dimers and half antibodies in samples before and after FAE under native conditions, the samples produced according to Example 2 (i.e., the reaction product and the unreacted control) were analyzed by HP-SEC (Agilent 1260 series) using TSK-gel G3000SWxl (Tosoh Bioscience-808541), TSK guard column SWXL (Tosoh Bioscience-808543), and HP-SEC buffer (200 mM sodium phosphate, 50 mM NaCl, pH 7.0; composed of sodium dihydrogen phosphate dihydrate (NaH2PO4, 2H2O; Sigma, ref. 71500), sodium 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.

[0299] The same amount of sample was injected (20 μg per sample, injection volume between 10 and 100 μL). The data obtained were used to estimate the purity in the sample (percentage of complete IgG dimers or half bodies). In order to ensure that the sample quality is sufficient, any starting material should contain more than 95% complete molecules. Otherwise, consider additional purification or repeated production. Based on the results at 280 nm, the retention time and relative peak area of ​​the peaks observed in the chromatogram were analyzed. The results are shown in Figure 3. The main peak shows IgG-IgG dimers, because if they are analyzed separately, there are no aggregates in the same sample.

[0300] After FAE, reactions containing CH3 variants with DE and KK (i.e., #1 and #3) detected a main peak corresponding to the size of IgG, with no aggregates but a small amount of half bodies. After FAE, reactions containing CH3 variants with 405L and 409R (i.e., #2 and #4) detected a main peak corresponding to the size of IgG, with no aggregates. After FAE of single single products (production) of samples obtained after FAE reactions with DE (#5 and #9) or KK (#6 and #10), CH3 variants mainly showed half bodies, while samples with 405L (#8 and #12) or 409R (#7 and #11) CH3 variants formed homodimers.

[0301] Example 6: Scaled-up FAE solution

[0302] Briefly, IgG molecules were incubated in the presence of β-mercaptoethylamine hydrochloride without shaking. Quantification was then performed using a Spark ALIAS autosampler equipped with a single HiPrep 26 / 10 desalting column (Cytiva). The Pure 25 system was used to exchange the sample buffer (Bex) into PBS. For reoxidation, the sample was kept at 4°C for a sufficient time to allow complete reoxidation to occur. The FAE reaction was performed on the IgG molecules produced in Example 1, as listed in Table 3.

[0303] Table 3. Expressed IgG antibodies used in FAE

[0304]

[0305]

[0306] Fresh MEA stock solution (750 mM) was prepared by dissolving 852 mg of cysteamine hydrochloride (Sigma catalog # 30078) in 5-6 mL of PBS (pH 7.4). The pH was adjusted to pH 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 a 750 mM stock solution and filtered with a 0.2 μm filter before use.

[0307] The IgG mixture listed in Table 3 was prepared at a final concentration of 1.1 mg / mL by adding PBS (using Fresenius Versylene sterile endotoxin-free water, product catalog number #B230531, diluted from a 10× stock solution Gibco, product catalog number #70011-051 pH 7.4) and adjusted to a final volume of 3.15 mL. 100 μL of this mixture was removed and kept at 4 degrees as an unreacted control, i.e., not exposed to MEA or Bex, and stored at 4°C in the dark until further use. To initiate FAE, 350 μL of 75 mM MEA was added to the remaining 3.05 mL, and the solution was gently mixed and kept at 31°C for 5 hours without shaking.

[0308] Buffer exchange:

[0309] After 5 hours, the samples were collected using a Cytiva ALIAS autosampler equipped with a HiPrep 26 / 10 desalting column (Cytiva catalog #17-5087-01). Pure 25 system, at a flow rate of 6mL / min and at 20 degrees Celsius, the sample buffer exchange (Bex) was exchanged to PBS (using Fresenius Versylene sterile endotoxin-free water, product catalog # B230531, diluted from 10× stock solution Gibco, product catalog # 70011-051 pH 7.4). For reoxidation, the sample was kept at 4°C for 64 hours (without further pipetting, shaking or mixing) to allow the material to fully reoxidize. For quality control purposes, 60-100 μg of each sample was retained before gel filtration purification (i.e., GF pre-control).

[0310] Gel filtration

[0311] 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 # UFC903096). The material was then loaded onto a Spark ALIAS autosampler equipped with Gel filtration purification was performed on a Pure 25 system using PBS as the mobile phase (PBS prepared above) to separate samples based on size using a Superdex 200 increase 16 / 40 column (Cytiva, catalog #29321905) at a flow rate of 1 mL / min (fraction size 0.5 mL, autosampler injection loop 10 mL, temperature 20°C). Samples were detected by UV light according to the manufacturer's instructions. The results of reactions #1-8 in Table 3 are shown in Figures 4a-4h, upper panels.

[0312] LabChip analysis

[0313] Fractions from the gel filtration column were analyzed on a LabChip GXII Touch instrument under non-reducing conditions to visualize the possible presence of IgG dimers and hemimers in the obtained gel filtration fractions. LabChip analysis was performed using HT Protein Expression Chips (Perkin-Elmer catalog #760499) and the Protein Clear HR Kit (Perkin Elmer CL8960014). LabChip analysis conditions: Input samples and fractions with a concentration 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. Sample analysis was performed using the standard "HT Protein Express" script according to the manufacturer's instructions using a LabChip GXII Touch instrument. The results of reactions #1-8 in Table 3 are shown in Figures 4a-4h, lower panel. Samples collected after FAE primarily contained IgG dimers. Few aggregates were observed. The asterisks for samples #1, #2, #5, and #6 indicate the IgG half-body peaks separated from the IgG dimers by gel filtration and LabChip analysis.

[0314] The fractions containing IgG dimers but not half bodies were pooled and the protein concentration of the pooled 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 the total protein program with a water blank and an extinction coefficient of 1.45 mL / mg / cm according to the manufacturer's instructions.

[0315] Pooled 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 #43819). Figure 5 As shown (non-reduced: upper panel, reduced: lower panel). The results from non-reduced conditions indicate that the purity of all IgG antibodies is at least 98%, and very small amounts of IgG with a single LC are observed. Therefore, using these samples, FAE does not cause a significant loss of light chains. The results from reduced conditions show that the dual-specific antibodies from reactions #1-4 and #5-8 share the same light chain mixture, which indicates the presence of two different Fab arms in the IgG samples.

[0316] In addition, "pre-GF control samples" for reactions #1-8 collected before and after FAE were analyzed by reducing and non-reducing LabChip analysis according to the procedures provided above. For all reactions, before the samples were subjected to FAE, they were confirmed to contain half-bodies, while after FAE, the samples showed fewer half-bodies (data not included).

[0317] Example 7: HP-CIEX analysis

[0318] To confirm that the FAE reaction resulted in the production of bispecific antibodies, all samples (reaction products and unreacted controls) produced in Example 2 were analyzed by CIEX (Agilent 1260 series). A chromatography column (TSK gel SP-STAT 7 μm, 4.6 mm ID × 10 cm L; Tosoh catalog #21964) was equilibrated with a low ionic strength phosphate buffer (buffer A) (25 mM sodium phosphate, pH 6.0 ± 0.05, consisting of sodium dihydrogen phosphate dihydrate (NaH2PO4, 2H2O; Sigma, ref. 71500) and sodium hydrogen phosphate dihydrate (Na2HPO4, 2H2O; Sigma, ref. 71643) dissolved in Milli-Q water, filtered through a 0.45 μm membrane filter). The antibody was then displaced from the column by increasing the Na+ concentration by running a gradient of phosphate buffer (Buffer B) (25 mM sodium phosphate, 1 M NaCl pH 6.0, composed of sodium phosphate monobasic dihydrate (NaH2PO4, 2H2O; Sigma, ref. 71500) and sodium phosphate dibasic dihydrate (Na2HPO4, 2H2O; Sigma, ref. 71643) in Milli-Q water, filtered through a 0.45 μm membrane filter) with increasing salt percentages. The injected sample mass for all test samples and controls was 10 μg, and the injection volume ranged from 10 to 100 μL. The peak shape, retention time, and peak area of ​​the main peak observed in the chromatogram were analyzed based on the results at 220 nm.

[0319] The results in the lower panels of Figure 6 show that the use of FAEs with DE / KK variants resulted in only about 1% homodimers (see reactions #1 and #5) or even undetectable amounts of homodimers (see reactions #2 and #6). The use of FAEs with variants 405L / 409R resulted in about 5-13% homodimers (see reactions #3, #4, #7, and #8).

[0320] Figure 6a and Figure 6c The results shown in the lower panels of the Figure 4 show the appearance of a peak that was not present before FAE, which accounted for approximately 87-89% or 95-96% of the material corresponding to the DEKK heterodimer eluted from CIEX (i.e., the peak at approximately 21 min for #1 and #2, and the peak at approximately 17 min for reactions #5 and #6).

[0321] Figure 6b and Figure 6dThe results shown in the lower panels of show the appearance of peaks not present prior to FAE, which account for approximately 77-84% or 83-88% of the material corresponding to the 405L / 409R heterodimer eluted from CIEX, respectively (i.e., peaks at approximately 21 minutes for reactions #3 and #4, and peaks at approximately 16 minutes for reactions #7 and #8). Figure 6a -d Asterisks represent peaks detected in each sample from #1 to #8, indicating that the appearance of these peaks is independent of the DE / KK or 405L / 409RFc forms used. Based on the position relative to the main heterodimer peak, it is expected that these peaks can be removed in a straightforward manner using standard separation techniques.

[0322] In summary, the results show that heterodimers can be produced on a larger scale than shown in Examples 1-5 and with unequal ratios of starting materials using either DE / KK or 405L / 409R substitutions. Gel filtration-purified samples with 405L / 409R substitutions contained 5-13% of the undesired homodimer, while in samples with DE / KK substitutions, the contaminant was more successfully removed, with a maximum reported level of 1% homodimer. Higher purities were achieved using DEKK-based substitutions compared to 405L / 409R substitutions (87-96% vs 77-88%, respectively).

[0323] Furthermore, the estimated total recovery of the dual-specific antibody in mg protein after FAE reaction was comparable between the two replacement systems, but samples subjected to FAE using DE / KK replacement were reported to be of higher purity.

[0324] It is noteworthy that the purified dual-specific antibodies produced by FAE were confirmed to still bind to their cognate antigens by ELISA. This showed that no binding to other targets occurred. All antibodies prepared by FAE showed specific binding to their targets, leading to the conclusion that despite the reduction and reoxidation of the Fab arms, antibody specificity was maintained after FAE. In addition, regardless of the DE:KK ratio selected before FAE, the dual-specific antibodies were found to bind similarly to each antigen.

[0325] sequence

[0326] SEQ ID NO.1

[0327] EVQLVQSGAEVKKPGASVKVSCKASGFTFTSYYIHWVRQAPGQGLEWIGWIYPENDNTKYNEKFKDRVTITADTSTSTAYLELSSLRSEDTAVYYCARDGYSRYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTKPPSREEMTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0328] SEQ ID NO.2

[0329] QVQLVESGGVVQPGGSLRLSCAASGFTFSNAWMHWVRQAPGKGLEWVAQIKDKSQNYATYVAESVKGRFTISRADSKNSIYLQMNSLKTEDTAVYYCRYVHYAAGYGVDIWGQGTTVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTKPPSREEMTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0330] SEQ ID NO.3

[0331] EVQLLEPGGGLVQPGGSLRLSCEASGSTFSTYAMSWVRQAPGKGLEWVSGFSGSGGFTFYADSVRGRFTISRDSSKNTLFLQMSSLRAEDTAVYYCAIPARGYNYGSFQHWGQGTLVTSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTDPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0332] SEQ ID NO.4EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIFPDDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKPSDTAMYYCVRLGGYSGYAEDFVDFWGQGTLVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHPCPPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTDPPSREEMTKNQVSLTCEVKGGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK

[0333] SEQ ID NO.5

[0334] EVQLVQSGAEVKKPGASVKVSCKASGFTFTSYYIHWVRQAPGQGLEWIGWIYPENDNTKYNEKFKDRVTITADTSTSTAYLELSSLRSEDTAVYYCARDGYSRYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0335] SEQ ID NO.6

[0336] QVQLVESGGVVQPGGSLRLSCAASGFTFSNAWMHWVRQAPGKGLEWVAQIKDKSQNYATYVAESVKGRFTISRADSKNSIYLQMNSLKTEDTAVYYCRYVHYAAGYGVDIWGQGTTVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0337] SEQ ID NO.7

[0338] EVQLLEPGGGLVQPGGSLRLSCEASGSTFSTYAMSWVRQAPGKGLEWVSGFSGSGGFTFYADSVRGRFTISRDSSKNTLFLQMSSLRAEDTAVYYCAIPARGYNYGSFQHWGQGTLVTSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0339] SEQ ID NO.8

[0340] EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIFPDDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKPSDTAMYYCVRLGGYSGYAEDFVDFWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0341] SEQ ID NO.9

[0342] DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWTSTRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0343] SEQ ID NO.10

[0344] DIVMTQSPLSLPVTPGEPASISCRSSQPLVHSNRNTYLHWYQQKPGQAPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0345] SEQ ID NO.11SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDNDRPSGLPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0346] SEQ ID NO.12

[0347] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

Claims

1. A method for producing 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 comprising positively charged amino acid residues at positions 351 and 366, and (b) a second protein comprising a second CH3 domain comprising negatively charged amino acid residues at positions 351 and 368, The numbering is based on EU numbering. - incubating the proteins of (a) and (b) together under reducing conditions to provide a reduced first protein and a reduced second protein; as well as - Reoxidizing the reduced protein 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 the group consisting of: a monomeric protein, a homodimeric protein, and a heterodimeric protein.

3. The method of 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: an antibody and a half antibody, or a fragment thereof, optionally wherein the antibody, half antibody or fragment thereof is a human antibody, half antibody or fragment thereof. The method of claim 4 , wherein the fragment is a monomeric Fc region or a dimeric Fc region.

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

7. The method of any one of the preceding claims, wherein the first protein is an antibody with a first binding specificity and the second protein is an antibody with 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 heterodimeric protein obtained is a heterodimeric antibody.

11. The method of claim 9 or 10, wherein the heterodimeric antibody is a multivalent antibody, optionally wherein the multivalent antibody is a bivalent, trivalent, tetravalent antibody or an antibody having up to six valencies.

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, trispecific or quadrispecific antibody.

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

14. The method of 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 of 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 of any one of the preceding claims, wherein the first CH3 domain comprises 351K and 366K, and the second CH3 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 of a) and b) is an Fc-engineered protein.

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

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 at position 235 and / or 236 that affects ADCC.

20. The method of any preceding claim, wherein the second protein is obtained independently of the first protein.

21. The method of any one of the preceding claims, wherein the reducing conditions comprise: (a) incubating the protein in the presence of a reducing agent, optionally wherein the reducing agent comprises or is selected from the group consisting of: 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) incubating the protein at a pH between 6.0 and 12.0; and / or (c) incubating 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 a step of enriching and / or isolating the heterodimeric protein obtained after reoxidation.

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

24. An isolated heterodimeric protein obtainable by the method of any preceding claim.

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, wherein the first CH3 domain comprises one or more of the amino acid variants 366K, 366R, 351K or 351R, and wherein the second CH3 domain comprises one or more of the amino acid variants 351E, 351D, 368E or 368D, The heterodimeric antibody also comprises two light chains with different sequences.

26. The isolated heterodimeric antibody of 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 of any one of claims 1-23, wherein the first protein and / or the second protein comprises an antibody binding domain, a 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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