Methods and means for production of ig-like molecules

Abstract

To provide heterodimeric proteins, and their methods of production and collection, that are useful to treat human disease.SOLUTION: Provided is a method for producing heterodimeric polypeptides such as Ig-like molecules, bispecific antibodies, or multivalent multimers. Also provided is an improved method for producing heterodimeric molecules from a mixture of CH3 domain polypeptides produced from a single cell.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to the fields of molecular biology, medicine and biological therapy, in particular to the field of therapeutic antibodies for the treatment of various diseases. [Background technology]

[0002] background In the past decade, bispecific antibodies have been presented as an alternative to the use of a combination of two antibodies. Similarly, multivalent multimers that can bind to three or more of the same or different antigens or epitopes have been presented as a developing technology. A combination of two antibodies is a mixture of two different immunoglobulins that bind to different epitopes on the same or different targets, whereas in bispecific antibodies, this is achieved by a single immunoglobulin. In multivalent multimers, binding to three or more different epitopes on the same or different targets can be achieved.

[0003] By binding to two epitopes on the same or different targets, bispecific antibodies can have similar or superior effects compared to a combination of two antibodies that bind to the same epitope. This can also be applied to the generation of multivalent multimers that can bind to three or more targets. Furthermore, the various effects of these formats have been observed as well, since IgG-type bispecific antibodies bind two different monovalent binding regions to a single molecule, a mixture of two IgG antibodies bind two different bivalent binding molecules to a single preparation, and multivalent multimers can bind three or more binding regions. From a technical and regulatory perspective, if these bispecific or multivalent multimers can be efficiently produced and separated in a substantially pure manner, the complexity of developing a single bispecific antibody or multivalent multimer is reduced, since a single molecule is involved in the manufacturing, preclinical trials, and clinical trials. Thus, therapies based on single bispecific antibodies or multivalent multimers are facilitated by a reduced complexity and cost-effective drug development process while providing more effective antibody therapies.

[0004] Bispecific antibodies based on the IgG format, consisting of two heavy chains and two light chains, have been produced in a variety of ways. For example, bispecific antibodies can be produced by fusing two antibody-secreting cell lines to create a new cell line or by expressing two antibodies in a single cell using recombinant DNA technology. These approaches can result in multiple antibody species, since each heavy chain of each antibody can form monospecific dimers (also called homodimers) containing two identical paired heavy chains with the same specificity, and bispecific dimers (also called heterodimers) containing two different paired heavy chains with different specificities, and unpaired heavy or light chains, called half bodies, can be a mixture of cell products. In addition, the light and heavy chains of each antibody can pair randomly to form non-functional combinations. This problem, known as heavy and light chain mispairing, can be addressed by selecting antibodies that share a common light chain for expression as bispecifics. Recently, applicants have shown that it is possible to generate multivalent multimers comprising three or more heavy chains and three or more light chains, either heavy or light chains, but preferably the light chains being a common chain.

[0005] However, even when a common light chain is used, expression of two or more heavy chains containing mutations that promote heterodimer formation and one common light chain in a single cell can result in three different antibody species and half-body impurities. Expression of heavy chains with engineered residue mutations in Fc may promote heterodimer formation but still generate remaining half-bodies, homodimers, or heterodimers that are less stable due to the engineered mutations. Expression of bispecific antibodies may result in expression of two monospecific "parent" antibodies and bispecific antibodies containing matching Fc regions. Following the formation of half-bodies and / or monospecific antibodies, the overall efficiency of the cell expression system may decrease, resulting in a more costly and time-consuming process to separate the desired bispecific antibody or multivalent multimer. Therefore, if a single bispecific antibody or multivalent multimer product is desired, the desired bispecific antibody or multivalent multimer needs to be purified from the resulting mixture.

[0006] The need to recover bispecific antibodies or multivalent multimers is important in the fields of preclinical, clinical and commercial production. Although techniques have been used to further increase the proportion of bispecific antibodies and reduce the proportion of mis-paired heavy and light chains in the mixture of parent and bispecific antibodies, there remains a need for bispecific and multivalent multimeric forms that eliminate or minimize some of the above-mentioned drawbacks and increase the amount of targeting moieties produced. Although techniques have been previously reported in the art to improve the heterodimerization pairing rate of bispecific or multivalent multimers, undesirable impurities may remain in the mixture of expression products. Furthermore, the separation of heterodimerization products, which are bispecific antibodies or multivalent multimers, can be difficult, inefficient and costly, depending on the separation means and the composition of the targeting moieties of interest.

[0007] Thus, there remains a need for improved and / or alternative techniques for producing and purifying biological therapeutics. Summary of the Invention

[0008] Disclosure Summary The present invention provides a novel means for producing and purifying biological therapeutics such as Ig-like molecules, bispecific antibodies and multivalent multimers. This provides a novel means for improving the interaction between at least two CH3 domains of interest when multiple CH3 domains are present in a mixture produced by, for example, a single cell (see, for example, WO2013157954 and WO2013157953).

[0009] Provided herein is an isolated heterodimeric protein comprising a first human IgG CH3-containing polypeptide and a second human IgG CH3-containing polypeptide, wherein said first human IgG CH3-containing polypeptide comprises amino acid mutations L351D and L368E, said second human IgG CH3-containing polypeptide comprises amino acid mutations T366K and L351K, and said first human IgG CH3-containing polypeptide comprises further amino acid mutations at positions S364, K409 and / or K360.

[0010] Suitably, the amino acid at position 364 may be valine, isoleucine, threonine, glutamine or leucine.

[0011] Suitably, the amino acid at position 409 may be isoleucine, leucine or glutamate.

[0012] Suitably, the amino acid at position 360 may be an aspartic acid.

[0013] Suitably, the heterodimeric protein may comprise the CH3 region of human IgG1, IgG2, IgG3 or IgG4.

[0014] Advantageously, the heterodimeric protein may comprise a human immunoglobulin Fc region. Optionally, the human immunoglobulin Fc region may comprise an IgG1, IgG2, IgG3 or IgG4 Fc region.

[0015] Suitably, the first CH3-containing polypeptide can be an antibody heavy chain.

[0016] Suitably, the second CH3-containing polypeptide can be an antibody heavy chain.

[0017] Suitably, the heterodimeric protein may further comprise one or more antibody light chains.

[0018] Suitably, the antibody light chain may be a common antibody light chain.

[0019] Pharmaceutical compositions comprising the isolated heterodimeric proteins described herein are also provided.

[0020] Also provided are isolated nucleic acids encoding the first and second human IgG CH3-containing polypeptides described herein.

[0021] Also provided are recombinant host cells comprising the isolated nucleic acids described herein.

[0022] Also provided is a method for producing an isolated heterodimeric protein as described herein, comprising culturing a recombinant host cell of the invention under conditions allowing expression of the first and second human IgG CH3-containing polypeptides.

[0023] Also provided is a method for improving the stability of a heterodimeric protein comprising a first human IgG CH3-containing polypeptide comprising the amino acid mutations L351D and L368E and a second human IgG CH3-containing polypeptide comprising the amino acid mutations T366K and L351K, wherein the method comprises introducing amino acid mutations into the first human IgG CH3-containing polypeptide at positions S364, K409 and / or K360.

[0024] Also provided is a method for reducing the stability of a homodimeric protein comprising a first human IgG CH3-containing polypeptide comprising amino acid mutations L351D and L368E, the method comprising introducing amino acid mutations at positions S364, K409 and / or K360 into the first human IgG CH3-containing polypeptide.

[0025] Also provided is a method for improving the yield of a heterodimeric protein comprising a first human IgG CH3-containing polypeptide and a second human IgG CH3-containing polypeptide, wherein said first human IgG CH3-containing polypeptide comprises amino acid mutations L351D and L368E and said second human IgG CH3-containing polypeptide comprises amino acid mutations T366K and L351K, comprising introducing amino acid mutations into the first human IgG CH3-containing polypeptide at positions S364, K409 and / or K360.

[0026] Also provided is a method for enhancing the purity of a heterodimeric protein comprising a first human IgG CH3-containing polypeptide and a second human IgG CH3-containing polypeptide, wherein said first human IgG CH3-containing polypeptide comprises amino acid mutations L351D and L368E and said second human IgG CH3-containing polypeptide comprises amino acid mutations T366K and L351K, the method comprising: (a) introducing amino acid mutations into the first human IgG CH3-containing polypeptide at positions S364, K409 and / or K360; (b) subjecting the heterodimeric protein to ion exchange chromatography.

[0027] Suitably, the ion exchange chromatography may be cation exchange chromatography.

[0028] Suitably, the amino acid at position 364 may be valine, isoleucine, threonine, glutamine or leucine.

[0029] Suitably, the amino acid at position 409 may be isoleucine, leucine or glutamic acid.

[0030] Suitably, the amino acid at position 360 may be an aspartic acid.

[0031] Suitably, the heterodimeric protein may comprise a human IgG1, IgG2, IgG3 or IgG4 CH3 region.

[0032] Suitably, the heterodimeric protein may comprise a human immunoglobulin Fc region. Optionally, the human immunoglobulin Fc region may comprise an IgG1, IgG2, IgG3 or IgG4 Fc region.

[0033] Suitably, the first CH3-containing polypeptide can be an antibody heavy chain.

[0034] Suitably, the second CH3-containing polypeptide can be an antibody heavy chain.

[0035] Suitably, the heterodimeric protein may further comprise one or more antibody light chains.

[0036] Suitably, the antibody light chain may be a common antibody light chain.

[0037] Also provided is a heterodimeric protein produced by the methods described herein.

[0038] Throughout the description and claims of this specification, the terms "comprise" and "contain" and variations thereof mean "including, but not limited to," and are not intended to exclude other moieties, additives, components, integers, or steps.

[0039] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating the plural as well as the singular.

[0040] It is to be understood that any feature, integer, property, compound, chemical moiety or group described in connection with a particular aspect, embodiment or example of the invention is applicable to any other aspect, embodiment or example described herein, except where not inconsistent therewith.

[0041] Various aspects of the invention are described in further detail below. [Brief description of the drawings]

[0042] BRIEF DESCRIPTION OF THE DRAWINGS In the discussion that follows, when a letter is followed by a number, this indicates the amino acid at that residue number position of the primary amino acid sequence according to EU numbering. For example, K351 indicates a lysine at position 351. When a letter is followed by a number and then another letter, this indicates the amino acid at the residue's native position according to EU numbering (e.g., wild type human Fc) and the residue that is engineered into that position (e.g., K409S indicates that a lysine is present at position 409 of the wild type Fc and the engineered variation is a serine).

[0043] Aspects of the invention are further described below with reference to the accompanying drawings.

[0044] [Figure 1] Figure 1: Vector map used for expression of heavy chain variants. [Diagram 2] Figure 2: SDS-PAGE results of single (A) and double (B) transfections of K360 variants. Band sizes seen on unreduced SDS-Page correspond to 150 KDa (IgG); 75 KDa (half-body); 50 KDa (heavy chain) and 25 KDa (light chain). [Diagram 3] Figure 3: SDS-PAGE results of single (A) and double (B) transfection of S364 variants. Band sizes seen on unreduced SDS-Page correspond to 150 KDa (IgG); 75 KDa (hemimeric); 50 KDa (heavy chain) and 25 KDa (light chain). [Figure 4]Figure 4: SDS-PAGE results of single and double transfection of K409 variants. Band sizes seen on unreduced SDS-Page correspond to 150 KDa (IgG); 75 KDa (half body); 50 KDa (heavy chain) and 25 KDa (light chain). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] The patents, scientific and technical literature cited in this specification establish the knowledge available to those skilled in the art at the time of filing. The entire disclosures of issued patents, published and pending patent applications, and other publications cited in this specification are incorporated herein by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the event of any discrepancies, the disclosures in this specification shall prevail.

[0046] Various aspects of the invention are described in further detail below.

[0047] Detailed Description The present invention provides methods for producing heterodimeric polypeptides, such as bispecific antibodies or multivalent multimers. It also provides improved methods for producing heterodimeric molecules from a mixture of CH3 domain polypeptides produced from a single cell. Corresponding heterodimeric proteins, compositions, nucleic acids and host cells are also provided herein. The present invention allows for the production of predominantly (and in certain embodiments, almost exclusively) heterodimeric polypeptides with high yields and a reduction in undesired species such as half-bodies or homodimers. The methods of the present invention are based on mutations in the CH3 interface of heterodimeric polypeptides to increase heterodimer stability and reduce homodimer formation. Details of the methods and compositions are provided herein.

[0048] The terms "heterodimer," "heterodimeric complex," or "heterodimeric molecule" are used interchangeably herein and refer to a molecule comprising at least a first polypeptide and a second polypeptide, where the second polypeptide differs in amino acid sequence from the first polypeptide by at least one amino acid residue. A heterodimer may include a "heterodimer" formed by a first and a second polypeptide, or may form a higher order tertiary structure in which polypeptides are present in addition to the first and second polypeptides.

[0049] Unless otherwise specified herein, the terms "first" polypeptide and "second" polypeptide, and variations thereof, are merely general identifiers and should not be construed as identifying a specific or a particular polypeptide or component of a heterodimer of the invention.

[0050] A variety of approaches have been described in the art to promote the formation of a specific antibody of interest, thereby reducing the content of undesired antibodies or by-products in the resulting mixture.

[0051] In the case of antibodies, it is well known that CH3-CH3 interactions are the main driver of Fc dimerization (Ellerson JR., et al., J. Immunol 1976 (116) 510-517; Deisenhofer J. biochemistry 1981 (20) 2361-2370). Furthermore, it is well known that when two CH3 domains interact with each other, they associate at a protein-protein interface that contains "contact" residues (also called contact amino acids, interface residues or interface amino acids). A contact amino acid of a first CH3 domain interacts with one or more contact amino acids of a second CH3 domain. The contact amino acids are generally within 5.5 Å (preferably within 4.5 Å) of each other in the three-dimensional structure of the antibody. The interaction between the contact residues from one CH3 domain and the contact residues from another CH3 domain may be, for example, van der Waals forces, hydrogen bonds, water-mediated hydrogen bonds, salt bridges or other electrostatic forces, attractive interactions between aromatic side chains, disulfide bonds, or other forces known to those skilled in the art. It has been previously shown that about one-third of the contact amino acid side chains in the human IgG1 CH3 domain interface may account for the majority of the contribution to the folding and association of the domain. It may further be assumed that other (adjacent) amino acid residues may affect the interaction at the protein-protein interface.

[0052] The approach of preventing the dimerization of antibody heavy chain has been used in the art.Many means of engineering CH3 domain to favor heterodimerization over homodimerization have been described in the art.The approach for producing a specific bispecific antibody of interest is based on the electrostatic engineering of contact residues in the CH3-CH3 interface.

[0053] For example, approaches for the production of predetermined bispecific antibodies or multivalent multimers of interest based on electrostatic manipulation of contact residues within the CH3-CH3 interface are described in U.S. Patent Nos. 9,248,182; 9,358,286; 9,248,182; and 9,758,805. Using a combination of computational modeling and iterative validation strategies, a new charge-to-charge pairing construct, termed "DEKK", was identified, which identifies contact residues by introducing altered charge polarity to favor electrostatic interactions that promote heterodimer formation while reducing charge-induced homodimerization. Notably, the L351D / T366K pair efficiently formed heterodimers and barely formed homodimers. Using further computational analysis, we introduced additional electrostatic interactions at the core of the heterodimer CH3 interface, generating the L351D, L368E / L351K, T366K variants or DEKK, which showed high yields of heterodimer formation. Furthermore, the levels of aggregation, unfolding, and in vivo half-life were similar to those of the native antibody. Structural analysis of the DEKK bispecific antibody revealed unexpected interactions. As predicted by computational modeling, residues Asp-351 and Glu-368 interacted with residue Lys-366 in the opposing CH3 domain. However, as a result of a local shift in the IgG backbone forming a stabilizing salt bridge interaction, residue Lys-351 did not interact with Asp-351 as expected, but formed interactions with residues Pro-352, Ser-354, and Glu-357. These interactions may explain the stability of the heterodimeric molecule, making it a very attractive platform for the development of novel bispecific antibodies.

[0054] The present invention is based on heterodimeric bispecific antibodies or multivalent multimers that contain mutated amino acid residues within the CH3 interface of the bispecific antibody or multivalent multimer, which can be used in combination with the previously described DEKK mutated amino acid residues, or in combination with other means of promoting heterodimerization previously described in the art, such as knob-in-hole or charge engineering of the Fc (CH2 and / or CH3 domains). For example, by using the above-described DEKK technology and including in a heavy chain that contains DE mutations and additional amino acid residue mutations at positions K360, S364 and / or K409, the efficiency with which heterodimeric pairings are generated can be further improved compared to homodimeric pairings (e.g., DEDE CH3 pairing).

[0055] Furthermore, in conditions of overexpression of one of the two heavy chain arms comprising the desired heterodimer, this can result in the generation of homodimers of such arms. For example, in some conditions, when DEKK heterodimer engineering is used, overexpression of the DE chain relative to the amount of the KK chain can result in the generation of DEDE homodimers. The use of mutations at positions K360, S364 and / or K409 (described herein) allows for destabilization of the homodimer, resulting in the generation of half-bodies or single heavy chain arms (e.g., DE chains rather than DEDE homodimers), allowing for simpler and more efficient purification steps and less interference in assays testing the molecule when present in a mixture with a bispecific antibody or multivalent multimer.

[0056] Additionally, production of half-bodies is preferred over homodimers because, in contrast to homodimers, they are more easily resolved in the protein batch used for testing by gel filtration. Furthermore, half-bodies tend to have greater peak separation in cation exchange chromatography (CIEX) compared to homodimer and heterodimer peaks, facilitating selection of retention relative to pure bispecific or multivalent multimers.

[0057] Another feature of one or more of the amino acid mutations described herein is that such mutations promote heterodimer formation, stability, and / or destabilize homodimers, thereby increasing the overall efficiency of the expression system.

[0058] In one aspect, the invention described herein provides a method of making a heterodimeric molecule (e.g., a bispecific antibody or a multivalent multimer) comprising two CH3 domains, where one CH3 domain comprises a CH3 variant residue at position K360, S364 and / or K409, said method comprising providing in a host cell: a first nucleic acid encoding a first CH3 domain-containing polypeptide chain, and a second nucleic acid encoding a second CH3 domain-containing polypeptide chain; wherein the first nucleic acid encodes one or more of the CH3 domain variant residues at positions K360, S364 and / or K409, and the method further comprises culturing the host cell to allow expression of the two nucleic acids and recovering the heterodimeric molecule from the culture.

[0059] In another aspect, the invention described herein provides a method of producing a heterodimeric molecule (e.g., a bispecific antibody or a multivalent multimer) comprising at least two different Ig-like molecules from a single host cell, each of said two Ig-like molecules comprising two CH3 domains capable of forming an interface, and wherein the CH3 domain of each heterodimeric molecule comprises one or more of the CH3 domain variant residues at positions K360, S364 and / or K409, said method comprising providing in the host cell: a) a first nucleic acid encoding a first CH3 domain-containing polypeptide chain; b) a second nucleic acid encoding a second CH3 domain-containing polypeptide chain; c) a third nucleic acid encoding a third CH3 domain-containing polypeptide chain, and d) a fourth nucleic acid encoding a fourth CH3 domain-containing polypeptide chain; wherein at least two of the nucleic acids comprise means for preferential pairing of the first and second CH3 domain-containing polypeptides with the third and fourth CH3 domains, wherein the nucleic acid encoding the first CH3 domain comprises a CH3 domain mutant residue at position K360, S364 and / or K409, and wherein the nucleic acid encoding the third CH3 domain comprises one or more CH3 domain mutant residues at positions K360, S364 and / or K409, and the method further comprises culturing the host cell to allow expression of the at least four nucleic acids and recovering the at least two different Ig-like molecules from the culture.

[0060] Another aspect includes the above invention, wherein the means of preferential pairing of said first and second CH3 domain containing polypeptides is different from the means of preferential pairing of said third and fourth CH3 domains.

[0061] In another aspect, the invention described herein provides a first nucleic acid encoding a first CH3 domain containing polypeptide chain further comprising a DE at position 351 and position 368, respectively, and a second nucleic acid encoding a second CH3 domain containing polypeptide chain comprising a KK at position 351 and position 366, wherein one or more of the CH3 domain mutant residues at positions K360, S364 and / or K409 are present on the first CH3 domain containing polypeptide chain and further comprising a DE at positions 351 and 368.

[0062] In another aspect, the invention described herein provides a first nucleic acid encoding a first CH3 domain-containing polypeptide chain that comprises a mutation that promotes heterodimerization with a second CH3 domain-containing polypeptide chain encoded by a second nucleic acid that comprises a complementary mutation that promotes heterodimerization, wherein the first nucleic acid further encodes one or more CH3 domain mutant residues (at positions K360, S364 and / or K409).

[0063] The phrase "cation exchange resin" refers to a solid phase that is negatively charged and has free cations for exchange with cations in an aqueous solution passing over or through the solid phase. The charge can be provided by attaching one or more charged ligands to the solid phase, e.g., by covalent bonding. Alternatively, or in addition, the charge can be an inherent property of the solid phase (e.g., as in the case of silica, which has an overall negative charge).

[0064] The term "host cell protein (HCP)" as used herein means a protein that is derived from the source of antibody production, i.e., the host cell, and is distinct from the recombinant cell product, such as a heterodimeric polypeptide (e.g., bispecific antibody or multivalent multimer). For bispecific antibodies that can be used as therapeutics, it is preferable to remove the HCP from the original antibody drug. As used herein, the expression "removed host cell protein" means to include all impurities except the product or target molecule of interest, e.g., the bispecific antibody or multivalent multimer being purified, and includes DNA and cell growth factors derived from the host cell in addition to the host cell proteins themselves. Thus, removing the host cell proteins can significantly increase the purity of the target molecule.

[0065] For example, it is often desirable to produce multiple heterodimeric proteins to more efficiently disrupt multiple biological pathways involved in a disease process or the invasion, replication and / or spread of a pathogen.

[0066] Mixtures of multiple heterodimeric proteins are also particularly useful for treating certain diseases. For example, tumor cells use various strategies to develop resistance during treatment with antibodies or small molecule drugs. Resistance may involve multiple cell surface receptors and soluble molecules, and it is believed to be beneficial to develop antibody-based therapies for cancer that simultaneously address multiple mechanisms associated with such diseases and evasion. When more than one mechanism associated with such diseases and evasion is involved, bispecific multivalent multimers or mixtures of heterodimeric proteins may provide an innovative and attractive therapeutic format. Preferably, such mixtures of heterodimeric proteins are produced by a single cell to facilitate a drug development process that is less complicated from a regulatory perspective and more cost-effective and feasible from a drug manufacturing and clinical development perspective. In a single cell-based approach, it is desirable to use methods that allow for the controlled and efficient production of bispecific antibodies, thus reducing or even completely eliminating the need to separate the desired mixture of bispecific IgG polypeptides from undesired monospecific IgG polypeptides. In the prior art, mixtures of monospecific and bispecific antibodies have been produced by a single cell (WO2004 / 009618), but these mixtures represent a complex mixture of several different bispecific and monospecific antibody species. A further object of the present invention is to provide means and methods for producing a defined mixture of heterodimeric proteins (e.g., bispecific antibodies) in a single cell. Preferably, methods are provided that result in a mixture of (bispecific) antibodies with a proportion of at least 95%, at least 97% or more than 99% of dimeric IgG polypeptides, regardless of the amount of monomeric by-products (see herein). Generally, in cells in which multiple intact IgG polypeptides are produced, there may be half-polypeptides (monomeric by-products) that can be easily removed by size exclusion chromatography as known in the art.

[0067] In one aspect, the invention provides a method for producing in a single cell a defined heterodimeric polypeptide (e.g., a bispecific antibody or a multivalent multimer) or a mixture of at least two different Ig-like molecules instead of a single (bispecific) antibody of interest. If the formation of other undesired dimeric antibody species is reduced or absent, the use of CIEX purification is impractical, preferably in the context of using high-throughput screening of expression products, and the presence of homodimers as opposed to half-bodies can significantly perturb the experimental protocol and distort the experimental results. The reduction or elimination of homodimers is achieved by introducing mutations in the CH3 interface (positions K360, S364 and / or K409). This can increase the repulsion of domains such as CH3 domains and increase the attraction of different CH3 domains.

[0068] These mutations can be introduced into the DE chain using DEKK CH3 technology. Alternatively, these mutations can be introduced with other heterodimerization techniques that promote preferential pairing of different heavy chains (heterodimerization) over pairing of identical heavy chains (homodimerization), such as knob-in-hole or charge engineering of the Fc (e.g., CH2 and / or CH3 domains). The resulting mixture is well-defined and its composition is controlled by the design of the CH3 domain mutations.

[0069] Furthermore, adjusting the expression level and / or the different transfection ratios used for expression will affect the composition of the mixture. In the method according to the invention, the first nucleic acid encodes a CH3 domain that preferentially pairs with the CH3 domain encoded by the second nucleic acid, or the first nucleic acid encodes a CH3 domain that preferentially pairs with the CH3 domain encoded by the second nucleic acid, and the third nucleic acid encodes a CH3 domain that preferentially pairs with the CH3 domain encoded by the fourth nucleic acid. The invention also provides a mixture of at least two different Ig-like molecules obtained by the method according to the invention.

[0070] As used herein, the term "preferential pairing of the first and second CH3 domain containing polypeptides" refers to obtaining a resulting dimer comprising a first CH3 domain containing polypeptide and / or a second CH3 domain containing polypeptide that is a dimer consisting of one first CH3 domain containing polypeptide paired with one second CH3 domain containing polypeptide, and in which the formation of dimers comprising a first CH3 domain containing polypeptide paired with another first CH3 domain containing polypeptide and a second CH3 domain containing polypeptide paired with another second CH3 domain containing polypeptide is restricted. Similarly, the term "preferential pairing of third and fourth CH3 domain-containing polypeptides" refers to obtaining dimers comprising a third CH3 domain-containing polypeptide and / or a fourth CH3 domain-containing polypeptide, in which the formation of dimers consisting of one third CH3 domain-containing polypeptide paired with one fourth CH3 domain-containing polypeptide is restricted to dimers comprising a third CH3 domain-containing polypeptide paired with another third CH3 domain-containing polypeptide and a fourth CH3 domain-containing polypeptide paired with another fourth CH3 domain-containing polypeptide. As a result, when a nucleic acid encoding a polypeptide comprising four different (A, B, C, D) CH3 domains is introduced into a single cell, a mixture of mainly two bispecific Ig-like molecules is generated instead of a mixture of ten different Ig-like dimers (AA, AB, AC, AD, BB, BC, BD, CC, CD and DD). Those skilled in the art using the variants described herein will understand that other desired combinations can also be generated using this technology.

[0071] In the method according to the invention, each of the above CH3 domain-containing polypeptide chains preferably further comprises a variable region or two or more variable regions that recognize a target epitope. The variable regions that are part of the CH3 domain-containing polypeptide chains are preferably shared and paired with a common light chain. In that case, only the VH of the variable regions are different, while the VL of all the variable regions are essentially the same. Thus, in one aspect, the method according to the invention is provided, which further comprises providing the host cell with a nucleic acid that encodes a common light chain. In one embodiment, each variable region of the CH3 domain-containing polypeptide chain recognizes a different target epitope. In one embodiment, each of the four variable regions of the four CH3 domains that comprise the polypeptide chain recognizes a different target epitope. For example, if the first nucleic acid encodes a heavy chain further comprising a variable domain having specificity for antigen A, the second nucleic acid encodes a heavy chain further comprising a variable domain having specificity for antigen B, the third nucleic acid encodes a heavy chain further comprising a variable domain having specificity for antigen C, and the fourth nucleic acid encodes a heavy chain further comprising a variable domain having specificity for antigen D, then a mixture is generated comprising a bispecific Ig-like molecule specific for AB and a bispecific Ig-like molecule specific for CD. The formation of monospecific antibodies (having AA, BB, CC or DD specificity) or bispecific antibodies having specificity for AC, AD, BC or BD may be reduced or even absent by means of preferential pairing of the domains comprising said first and second CH3 domain-containing polypeptides and said third and fourth CH3 domain-containing polypeptides. This occurs when different heterodimerization procedures are used for the first and second CH3 domain-containing polypeptides and the third and fourth CH3 domain-containing polypeptides. Of course, it is also possible to use additional nucleic acids encoding, for example, fifth and sixth CH3 domain-containing polypeptides to generate defined mixtures containing two or more different Ig-like molecules (e.g., AB and CD).

[0072] Alternatively, a heavy chain comprising variable domains with specificity for antigens A and B as described above may comprise additional variable domains with specificity for antigens C or D linked via a linker, thereby forming a multivalent multimer, where heavy chains A and B may dimerize via a heterodimerization engineering such as DEKK, and homodimers are reduced by including the above mutations in one heavy chain to destabilize homodimer formation and generate half-bodies.

[0073] Of note, the ratio of nucleic acids used in the method according to the invention does not have to be 1:1:1:1, and the ratio of the resulting Ig-like molecules expressed does not have to be 1:1. It is possible to use means known in the art to generate a mixture of antibodies in an optimized ratio. For example, the expression level of the nucleic acids, and thus the ratio of the resulting Ig-like molecules produced, can be regulated by using various genetic elements such as promoters, enhancers, repressors, or by controlling the copy number of the DNA construct encoding the antibody, the site of genomic integration. Preferably, the amount of the expressed first human IgG CH3-containing polypeptide, comprising amino acid mutations L351D and L368E and mutated residues at positions S364, K409 and / or K360, is higher than the amount of the second human IgG CH3-containing polypeptide with amino acid mutations T366K and L351K. Preferably, the weight ratio of the first human IgG CH3-containing polypeptide to the second human IgG CH3-containing polypeptide is between 1:1 and 10:1. More preferably, the amount of DE-containing polypeptide is between 1:1 and 5:1, more preferably between 1.1 and 3:1, or between 1:1 and 2:1.

[0074] One aspect of the invention provides a method according to the invention, wherein said means for preferential pairing of said first and second CH3 domain-containing polypeptides is different from said means for preferential pairing of said third and fourth CH3 domain-containing polypeptides. By "different" it is meant that the means for preferential pairing of the first and second CH3 domain-containing polypeptides is designed to favor preferential pairing of the first and second chains. The design is such that essentially no interaction occurs between the first and third and / or fourth CH3 domain-containing polypeptide chains. In other words, dimerization between said first CH3 domain-containing polypeptide and said third or fourth polypeptide is reduced to essentially zero, etc. The third and fourth CH3 domain-containing polypeptides may be wild type or may comprise means for preferential pairing that are different from the means for preferential pairing of the first and second CH3 domains.

[0075] The present invention provides a method for efficient and controlled production of well-defined mixtures of Ig-like molecules with a high percentage of bispecific antibodies in the mixture. A percentage of (two) bispecifics of at least 95%, at least 97%, or more is obtained in systems where two bispecifics are desired. This means that only a maximum of 5%, and at most 3%, of monospecific bivalent by-products is obtained. It is noted that the amount of monomeric by-product half-molecules is not critical, since these half-molecules are easily separated from dimers by utilizing their size difference and cannot cross-link receptors, for example. Thus, the invention described herein provides the use of CH3 variants in the heavy chain arms that, upon expression of the bispecific antibody, reduce the production of homodimers in favor of half-bodies while maintaining the stability of the bispecific antibody, facilitating the purification of the bispecific and reducing homodimers that interfere with high-throughput screening and assaying of the desired bispecific antibody. Preferably, the variants of the invention provided in the CH3 domain reduce the amount of homodimeric by-products, enhance the formation of heterodimeric target molecules, and reduce the amount of half-bodies produced, thereby increasing the overall efficiency of the expression system and facilitating downstream separation of the target molecule from the HCP.

[0076] In another embodiment, the variable regions of the first and second CH3 domains comprising polypeptide chains recognize different target epitopes, but the variable regions of the third and fourth CH3 domains comprising polypeptide chains recognize the same target epitope. This mainly produces one bispecific Ig-like molecule and one monospecific Ig-like molecule. For example, when the variable regions of the first and second CH3 domains comprising polypeptide chains recognize different target epitopes, and when the variable regions of the third and fourth CH3 domains comprising polypeptide chains both recognize the same target epitope that is different from the target epitopes recognized by the first and second CH3 domains, a mixture of Ig-like molecules with specificity for AB or CC is formed. Thus, the method according to the present invention is further provided, in which the target epitopes recognized by the variable regions of the third and fourth CH3 domains comprising polypeptide chains are the same, but different from the target epitopes recognized by the variable regions of the first or second CH3 domains comprising polypeptide chains.

[0077] Alternatively, when the variable regions of the first and second CH3 domains comprising a polypeptide chain recognize different target epitopes, and when the variable regions of the third and fourth CH3 domains comprising a polypeptide chain both recognize the same epitope as the first or second CH3 polypeptide comprising a polypeptide chain, a mixture of Ig-like molecules with specificity for AB and AA, or AB and BB, is formed.Therefore, the method according to the present invention is also provided herein, in which the target epitopes recognized by the variable regions of the third and fourth CH3 domains comprising a polypeptide chain are the same as the target epitopes recognized by the variable regions of the first or second CH3 domains comprising a polypeptide chain.

[0078] In some embodiments, the heterodimeric polypeptide of the present invention is an immunoglobulin-like (Ig-like) polypeptide. The term "Ig-like polypeptide" as used herein refers to a protein moiety having at least one immunoglobulin (Ig) domain. The Ig-like polypeptide comprises a sequence that comprises at least the function of an immunoglobulin CH3 domain, preferably the sequence comprises an IgG1 CH3 domain. The protein moiety having at least a CH3 domain may further comprise a specific binding moiety. Thus, the CH3 domain of the present invention comprising a means for preferential pairing can be used for preferential pairing of two CH3 domains comprising protein moieties to design a desired heterodimeric binding molecule or mixture of binding molecules. The binding moiety that can be engineered into the CH3 domain containing protein moiety can be any binding agent, including but not limited to single chain Fv, single chain or tandem diabodies (TandAb®), VHH, Anticalins®, Nanobodies®, BiTE®, Fab, ankyrin repeat proteins or DARPIN®, Avimers®, DART, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins® or KALBITOR®. In one embodiment, the binding moiety is an antibody variable region (VH / VL combination). The variable regions that are part of a CH3 domain containing polypeptide chain preferably share a common light chain. In that case, only the VH of the variable regions differs, while the VL of all variable regions is essentially the same.

[0079] Alternatively, or in addition, other polypeptides, including cytokines, hormones, soluble ligands, receptors and / or peptides, can be engineered to the CH3 domains of the invention.

[0080] In one embodiment, the Ig-like polypeptide comprises a full-length Fc scaffold. In another embodiment, the Ig-like polypeptide is an antibody. The variable regions of these antibodies preferably share a common light chain, but may differ in their VH regions. The term "antibody" as used herein means a protein molecule belonging to the immunoglobulin class of proteins and comprises one or more domains that bind to an epitope on an antigen, such domains being derived from or sharing sequence homology with the variable region of the antibody. Antibodies are known in the art and include several isotypes, such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE and IgM. Antibodies according to the invention may be any of these isotypes, or functional derivatives and / or fragments thereof. In one embodiment, Ig-like molecules are produced that are antibodies of the IgG, IgA, IgD, IgE or IgE isotype. In one embodiment, Ig-like molecules are produced that are antibodies of the IgG isotype, since IgG antibodies have, for example, a longer half-life compared to antibodies of other isotypes.

[0081] The antibodies produced by the method according to the invention may have sequences of any origin, including mouse, avian, such as chicken, and human sequences. An antibody may be composed of sequences from only one origin, such as a fully human antibody, or may have sequences from two or more origins, such as a chimeric or humanized antibody. The binding of an antibody can be expressed in terms of specificity and affinity. Specificity determines which antigen or epitope is bound by a binding domain. Affinity is a measure of the strength of binding to a particular antigen or epitope. As used herein, the term "antigen" refers to a substance or molecule that can be bound by the antigen-binding site of an antibody. Also, a mixture of antigens can be considered as an "antigen". One skilled in the art will appreciate that a tumor cell lysate or a virus particle may be referred to as an "antigen", but in such a tumor cell lysate or virus particle preparation, many antigenic determinants are present. An antigen comprises at least one, and often more than one, epitope. As used herein, the term "epitope" refers to the part of an antigen that is recognized by the immune system, specifically antibodies, B cells, or T cells.

[0082] The term "CH3 domain" is well known in the art. There are four chains in the IgG structure: two light chains and two heavy chains, each light chain has two domains, a variable light chain and a constant light chain (VL and CL), and each heavy chain has four domains, a variable heavy chain (VH) and three constant heavy chain domains (CH1, CH2, CH3). The CH2 and CH3 domain regions of the heavy chains are called the Fc (fragment crystallizable) portion, Fc fragment, Fc scaffold, or simply Fc. An IgG molecule is a heterotetramer with two heavy chains linked by disulfide bonds (-SS-) at the hinge region, and two light chains linked to the heavy chains via -SS- disulfide bonds. The heavy chains dimerize through interactions at the CH3-CH3 domain interface and interactions at the hinge region, etc. The number of hinge disulfide bonds varies depending on the immunoglobulin subclass (Papadea and Check 1989). The Fc fragment of an immunoglobulin molecule is a dimer of two C-terminal constant regions of the heavy chain, the CH2 and CH3 domains. Its physiological functions include interaction with the complement system and with specific receptors on the surface of various cells. It is known that the interaction between the CH3 domains of two individual heavy chains plays an important role in promoting the dimerization of the heavy chains. Thus, the CH3 domain directs the binding of antibody heavy chains (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). Thus, the CH3 variants of the present invention can be used in combination with other antibody domains to generate full-length antibodies that are either bispecific or monospecific. The specificity of an antibody defined by the VH / VL combination does not normally affect the dimerization behavior of the heavy chain, which is driven by the CH3 domain.

[0083] The contact residues in the CH3-CH3 interface are either charged or neutral amino acid residues at physiological conditions. As used herein, the term "charged amino acid residue" or "charged residue" refers to amino acid residues that have a charged side chain. These are either the positively charged side chains that can be found in arginine (Arg, R), histidine (His, H) and lysine (Lys, K) or the negatively charged side chains that can be found in aspartic acid (Asp, D) and glutamic acid (Glu, E). As used herein, the term "neutral amino acid residue" or neutral residue refers to all other amino acids that do not have a charged side chain. These neutral residues include serine (Ser, S), threonine (Thr, T), asparagine (Asn, N), glutamine (Glu, Q), cysteine ​​(Cys, C), glycine (Gly, G), proline (Pro, P), alanine (Ala, A), valine (Val, V), isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, T).

[0084] The term "CH3-CH3 domain interface", or "CH3 interface", "CH3-CH3 pairing", "domain interface", or simply "interface", as used herein, refers to the association between two CH3 domains of separate CH3 domains. It includes polypeptides that are the result of interactions of amino acid residues, including at least one interaction between an amino acid of a first CH3 domain and an amino acid of a second CH3 domain. Such interactions may be due to, for example, van der Waals forces, hydrogen bonds, water-mediated hydrogen bonds, salt bridges or other electrostatic forces, attractive interactions between aromatic side chains, formation of disulfide bonds, or other forces known to those of skill in the art.

[0085] The invention further provides a method of making a host cell for producing at least two different Ig-like polypeptides, comprising the step of introducing into said host cell nucleic acid sequences encoding at least a first and a second CH3 domain-containing polypeptide, wherein at least two of said nucleic acid sequences provide a means for preferential pairing of said first and second CH3 domain-containing polypeptides, said nucleic acid sequences being introduced sequentially or simultaneously. Similarly, the host cell may already have nucleic acid sequences encoding at least a first and a second CH3 domain-containing polypeptide chain introduced into said host cell, wherein the nucleic acid encoding the variable regions is integrated adjacent to the nucleic acid encoding the CH3 domain, such that the host cell can be used as a master cell for integration of different variable regions for the production of different heterodimeric molecules, wherein at least two of said nucleic acid sequences encoding at least a first and a second CH3 domain provide a means for preferential pairing of said first and second CH3 domain-containing polypeptides.

[0086] A further aspect of the invention is to provide a method of making a host cell for producing a heterodimeric polypeptide, comprising the step of introducing into said host cell nucleic acid sequences encoding at least a first and a second CH3 domain-containing polypeptide chain, wherein said first CH3 domain-containing polypeptide chain comprises a positively charged amino acid residue and a wild-type CH3 generally has a neutral amino acid residue at that position, and said second CH3 domain-containing polypeptide chain comprises a negatively charged amino acid and a wild-type CH3 generally has a neutral amino acid residue at that position, said nucleic acid sequences being introduced sequentially or simultaneously. Preferably, said method of making said host cell further comprises the step of introducing into said host cell a nucleic acid sequence encoding a common light chain, or alternatively, said method further comprises the step of transiently, but preferably stably, integrating into a host cell already carrying a nucleic acid sequence encoding a common light chain.

[0087] Also provided herein is a recombinant host cell comprising a nucleic acid sequence encoding at least a first, second, third and fourth CH3 domain comprising a polypeptide chain, at least two of said nucleic acids comprising means for preferential pairing of the first and second CH3 domain containing polypeptides and said third and fourth CH3 domain containing polypeptides.

[0088] The invention further provides a recombinant host cell comprising a nucleic acid sequence encoding at least a first and a second CH3 domain-containing polypeptide chain, wherein said first CH3 domain-containing polypeptide chain comprises a positively charged amino acid residue at a position where a wild-type CH3 normally has a neutral amino acid residue, and said second CH3 domain-containing polypeptide chain comprises a negatively charged amino acid residue at a position where a wild-type CH3 normally has a neutral amino acid residue.

[0089] A recombinant host cell according to the invention may further comprise nucleic acid encoding a common light chain.

[0090] A "host cell" according to the present invention may be any host cell capable of expressing a recombinant DNA molecule, for example bacteria such as E. coli, Enterobacter, Salmonella, Bacillus, Pseudomonas, Streptomyces, yeasts such as Saccharomyces cerevisiae, K. lactis, P. pastoris, Candida or Yarrowia, filamentous fungi such as Neurospora, Aspergillus oryzae, Aspergillus nidulans and Aspergillus niger, insect cells such as Spodoptera frugiperda SF-9 or SF-21 cells, and preferably mouse cells including Chinese hamster ovary (CHO) cells, BHK cells, SP2 / 0 cells and NS-0 myeloma cells, primate cells such as COS and Vero cells, MDCK cells, BRL 3 cells, and the like. The cells may be A cells, hybridomas, tumor cells, immortalized primary cells, human cells such as W138, HepG2, HeLa, HEK293, HT1080, or embryonic retinal cells such as PER. C6. In many cases, the optimal expression system may include a mammalian cell expression vector and host so that the antibody can be properly glycosylated. Human cell lines can be used to obtain antibodies with a completely human glycosylation pattern. Conditions for cell growth or proliferation (see, for example, Tissue Culture, Academic Press, Kruse and Paterson, editors (1973)) and conditions for expression of recombinant products may vary somewhat, and optimization of the process is usually performed to increase the ratio and / or proliferation of cells relative to each other, according to methods commonly known to those skilled in the art. In general, principles, protocols, and practical techniques for maximizing productivity of mammalian cell culture are described in Mammalian Cell Biotechnology: a Practical Approach (ed. M. Butler, IRL Press, 1991). The expression of antibodies in recombinant host cells has been widely described in the art. The nucleic acids encoding the light and heavy chains can be present as extrachromosomal copies and / or stably integrated into the host cell chromosomes.

[0091] A further aspect of the present invention provides a culture of a recombinant host cell according to the invention, or obtainable or obtained by a method according to the invention, wherein said culture produces one or more heterodimeric polypeptides.

[0092] It is well known to those skilled in the art that in order to obtain expression of a nucleic acid sequence encoding a CH3 domain-containing polypeptide, a sequence capable of driving such expression can be functionally linked to the nucleic acid sequence encoding the CH3 domain-containing polypeptide. Functionally linked means that the nucleic acid sequence encoding the CH3 domain-containing polypeptide or its precursor is linked to a sequence capable of driving expression, allowing these sequences to drive the expression of the CH3 domain-containing polypeptide or its precursor. Useful expression vectors are available in the art, for example, Invitrogen's pcDNA vector series. When the sequence encoding the polypeptide of interest is properly integrated with respect to sequences that govern the transcription and translation of the encoded polypeptide, the resulting expression cassette is useful for producing the polypeptide of interest, referred to as expression. Sequences that drive expression can include promoters, enhancers, etc., and combinations thereof. These must be capable of functioning in the host cell, thereby driving the expression of the nucleic acid sequence functionally linked to them. Promoters can be constitutive or regulatable, and can be obtained from a variety of sources, including viral, prokaryotic or eukaryotic sources, or can be artificially designed. The expression of the nucleic acid of interest can be from the native promoter or its derivatives, or from a completely heterologous promoter. Some well-known and widely used promoters for expression in eukaryotic cells are promoters derived from viruses such as adenovirus, such as E1A promoter, promoters derived from cytomegalovirus (CMV), such as CMV immediate early (IE) promoter, Simian Virus 40 (SV40), etc. Suitable promoters may be derived from eukaryotic cells, such as metallothionein (MT) promoter, elongation factor 1a (EF-1a) promoter, actin promoter, immunoglobulin promoter, heat shock promoter, etc. Any promoter or enhancer / promoter capable of driving the expression of a sequence of interest in a host cell is suitable in the present invention.In one embodiment, the sequence capable of driving expression comprises a region derived from the CMV promoter, preferably comprising nucleotides -735 to +95 of the CMV immediate early gene enhancer / promoter. The skilled artisan will recognize that the expression sequences used in the present invention may be appropriately combined with elements capable of stabilizing or enhancing expression, such as insulators, matrix attachment regions, STAR elements (WO03 / 004704), etc. This may improve the stability and / or level of expression.

[0093] Protein production in recombinant host cells has been extensively described, for example, in Current Protocols in Protein Science, 1995, Coligan JE, Dunn BM, Ploegh HL, Speicher DW, Wingfield PT, ISBN 0-471-11184-8; Bendig, 1988. Culturing of cells is performed to allow the cells to metabolize and / or grow and / or divide and / or produce the recombinant protein of interest. This can be accomplished by methods well known to those skilled in the art, including, but not limited to, providing nutrients to the cells. Methods include growth attached to a surface, growth in suspension, or a combination thereof. To optimize protein production yield, some culture conditions can be optimized by methods well known in the art. Cultivation can be performed, for example, in dishes, flasks, roller bottles, or bioreactors, using batch, fed-batch, continuous systems, hollow fibers, etc. To achieve large-scale (continuous) production of recombinant proteins by cell culture, cells capable of growing in suspension are used, and the cells can be cultured in the absence of animal or human serum or animal or human serum components, thus making purification easier and safer due to the absence of additional animal or human proteins from the culture medium, and also making the system highly reliable, since synthetic media are most reproducible.

[0094] Immunoglobulin-like polypeptides are expressed in host cells and harvested from cells or preferably from cell culture medium by methods generally known to those skilled in the art. After harvesting, these Ig-like polypeptides can be separated using methods known in the art. Such methods can include precipitation, centrifugation, filtration, size exclusion chromatography, affinity chromatography, cation and / or anion exchange chromatography, hydrophobic interaction chromatography, etc. For antibody mixtures that contain IgG polypeptides, Protein A or Protein G affinity chromatography can be suitably used (see, for example, U.S. Patent Nos. 4,801,687 and 5,151,504).

[0095] Following capture using affinity chromatography, an orthogonal polishing step is used to remove remaining process-related impurities that may include homodimers, charge variants, HCPs and DNA. In general, to obtain isolated bispecific antibodies or multivalent multimers, steps such as host cell culture, harvest clarification, followed by protein capture, anion exchange chromatography including removal of host cell DNA, followed by removal of host cell proteins, leached protein A and potential aggregates using CIEX, followed by additional steps such as virus filtration. Those skilled in the art will be aware that the order of such steps may be changed or individual steps may be substituted or removed. For example, alternatives to the second polishing step include hydrophobic interaction chromatography and mixed mode chromatography.

[0096] The immunoglobulin-like polypeptides and / or mixtures thereof produced by the method according to the invention preferably have a common light chain. Thus, a method according to the invention is further provided, further comprising providing said host cell with a nucleic acid encoding a common light chain. This is a light chain that can pair with at least two different heavy chains, thereby forming a functional antigen-binding domain. A functional antigen-binding domain can specifically bind to an antigen. In one embodiment, a common light chain is used that can pair with all heavy chains produced by the method according to the invention, thereby forming a functional antigen-binding domain, thus avoiding mispairing of mismatched heavy and light chains. In one aspect, only a common light chain with one identical amino acid sequence is used. Alternatively, the skilled artisan will recognize that "common" also means functional equivalents of light chains that do not have identical amino acid sequences. There are many variants of said light chains, in which there are mutations (deletions, substitutions, additions) that do not substantially affect the formation of a functional binding region. Thus, such variants can also bind to different heavy chains and form functional antigen-binding domains. Thus, the term "common light chain" as used herein refers to a light chain that may be identical or have some amino acid sequence differences while retaining the binding specificity of the resulting antibody after pairing with the heavy chain. For example, it is possible to prepare or find a non-identical but functionally equivalent light chain by introducing and testing conservative amino acid mutations and / or amino acid mutations in regions that do not contribute or only partially contribute to the binding specificity, such as pairing with the heavy chain. A combination of a particular common light chain and such functionally equivalent variants is encompassed by the term "common light chain". For a detailed description of the use of common light chains, see WO2004 / 009618. Preferably, a common light chain is used in the present invention that is a germline-like light chain, more preferably a germline light chain, preferably a rearranged germline human kappa light chain, most preferably a rearranged germline human kappa light chain IgVκ1-39 / Jκ or IGVκ3-20 / Jκ. The common light chain preferably comprises the light chain variable region described above with 0 to 5 amino acid insertions, deletions, substitutions, additions or combinations thereof. Another preferred common light chain is the human κ light chain IgVκ1-39 / IGJκ5.Preferably, the antibody of the present invention comprises the variable region of human κ light chain IgVκ1-39 / IGJκ5. A more preferred common light chain is human κ light chain IgVκ3-15 / IGJκ1. Preferably, the antibody of the present invention comprises the variable region of human κ light chain IgVκ3-15 / IGJκ1. A more preferred common light chain is human κ light chain IgVκ3-20 / IGJκ1. Preferably, the antibody of the present invention comprises the variable region of human κ light chain IgVκ3-20 / IGJκ1. A more preferred common light chain is human λ light chain IgVλ3-21 / IGJλ3. Preferably, the antibody of the present invention comprises the variable region of human κ light chain IgVλ3-21 / IGJλ3.

[0097] Alternatively, instead of using a common light chain and to avoid mispairing of mismatched heavy and light chains, the skilled artisan can choose means for forced pairing of heavy and light chains, e.g. as described in WO2009 / 080251, WO2009 / 080252 and / or WO2009 / 080253.

[0098] The inventors have previously described the expression of CH3 amino acids where the wild type CH3 has a charged residue with a neutral amino acid residue at that position, and the alternative charges of the two CH3 domains cause a stable interaction (e.g., a first CH3 with positively charged residues at positions 351 and 366, and a second CH3 with negatively charged residues at positions 351 and 368). The variations and modified heavy chains of the present invention can be incorporated with the above means of promoting heterodimerization, or other means known to those skilled in the art. The invention described herein provides the additional ability to promote heterodimerization of the CH3 domains, and may increase the stability of heterodimerization or decrease the stability of homodimers based on amino acid residue variants at the CH3 interface, and preferably have a combination of the above advantages.

[0099] The heterodimers of the invention are more stable and easier to separate from mixtures and impurities compared to wild-type dimers (wild-type dimers are defined as bispecific IgG (AB) without CH3 engineering, in contrast to their homodimers (AA or BB)). Surprisingly, it has become possible to further increase the percentage of one or more Ig-like polypeptides of interest in a mixture. Methods known in the art for the preferential production of heterodimers (e.g. bispecific antibodies) generally involve the production of several undesirable dimeric by-products. For example, the percentage of bispecific antibodies of interest using the knob-into-hole technique known to those skilled in the art is reported to be at most 87%, whereas electrostatic engineering approaches, in which charged contact amino acids are replaced by the opposite amino acids, also reportedly result in percentages of up to 96%. The inventors have succeeded in introducing mutations that further increase the percentage of heterodimeric polypeptides of interest.

[0100] The present invention relates to a CH3 domain comprising at least one mutated residue at positions K360, S364 and / or K409. These mutated residues are described in more detail below. A letter followed by a number followed by another letter indicates the amino acid at the original position of the residue according to EU numbering (e.g., wild type human Fc) and the engineered residue at that position (e.g., K409S indicates that a lysine is present at position 409 in wild type Fc. The engineered mutation is a serine). For EU numbering, see http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html.

[0101] One aspect of the invention provides a method for producing a heterodimeric polypeptide from a single cell, wherein said heterodimeric polypeptide comprises amino acid mutations at positions K360, S364 and / or K409, said method comprising providing said cell with: a. a first nucleic acid encoding a first CH3 domain-containing polypeptide chain; b. a second nucleic acid encoding a second CH3 domain-containing polypeptide chain; wherein the first CH3 domain-containing polypeptide comprises a mutation at position K360, S364 and / or K409, and the method further comprises culturing the host cell to allow expression of the two nucleic acids, and recovering the heterodimeric Ig-like polypeptide from the culture.

[0102] Thus, one aspect of the invention provides a heterodimeric polypeptide comprising two CH3 domains, wherein said first CH3 domain-containing polypeptide chain comprises a DE amino acid mutation and said second CH3 domain-containing polypeptide chain comprises a KK amino acid mutation, wherein one or more residues at one or more positions K360, S364 and / or K409 in said first CH3 domain-containing polypeptide chain are mutant compared to the human wild-type residue.

[0103] One aspect of the invention provides a method of producing a heterodimeric polypeptide from a single cell comprising providing a heterodimeric polypeptide to a single cell, said method comprising providing in the cell: a. a first nucleic acid encoding a first CH3 domain-containing polypeptide chain; b. a second nucleic acid encoding a second CH3 domain-containing polypeptide chain; wherein the first CH3 domain-containing polypeptide chain comprises DE and further amino acid mutations at positions K360, S364 and / or K409, wherein the second CH3 domain-containing polypeptide chain comprises a KK, The method further comprises the steps of culturing the host cell to allow expression of the two nucleic acids, and recovering the heterodimeric Ig-like polypeptide from the culture.

[0104] In another embodiment, the mutated residues at positions K360, S364 and / or K409 are combined with other heterodimerization variations including, for example, knob-into-hole, reverse charge substitutions in the CH3 region, or other techniques known to those of skill in the art.

[0105] In one aspect, the method further comprises the step of providing said host cell with a nucleic acid encoding a common light chain, which has the advantages outlined herein above.

[0106] As described elsewhere herein, a heterodimeric protein is described that comprises a first CH3-containing polypeptide and a second CH3-containing polypeptide, wherein the first CH3-containing polypeptide comprises additional amino acid mutations at positions S364, K409 and / or K360. Optionally, the first and second CH3 polypeptides are of human origin.

[0107] For example, the first CH3-containing polypeptide described herein can include the S364V amino acid.

[0108] Alternatively, the first CH3-containing polypeptide described herein can include an S364I amino acid.

[0109] In another example, the first CH3-containing polypeptide described herein can include an S364T amino acid.

[0110] In a further example, the first CH3-containing polypeptide described herein can include an S364Q amino acid.

[0111] Alternatively, the first CH3-containing polypeptide described herein can include the S364L amino acid.

[0112] The first CH3-containing polypeptide described herein may comprise an amino acid mutation at position K409 instead of, or in addition to, an amino acid mutation at position S364 (such as, for example, S364V, S364I, S364T, S364Q or S364L, as detailed above).

[0113] Thus, the first CH3-containing polypeptide described herein may optionally include an amino acid mutation at position S364 (e.g., S364V, S364I, S364T, S364Q or S364L, as detailed above), as well as a K409I amino acid.

[0114] Alternatively, the first CH3-containing polypeptide described herein may optionally include an amino acid mutation at position S364 (e.g., S364V, S364I, S364T, S364Q or S364L, as detailed above), as well as a K409L amino acid.

[0115] In a further example, the first CH3-containing polypeptide described herein may optionally include an amino acid mutation at position S364 (e.g., S364V, S364I, S364T, S364Q or S364L, as detailed above), as well as a K409E amino acid.

[0116] The first CH3-containing polypeptide described herein may comprise an amino acid mutation at position K360 instead of, or in addition to, an amino acid mutation at position S364 (such as, for example, S364V, S364I, S364T, S364Q or S364L, as detailed above) and / or a mutation at position K409 (such as, for example, K409I, K409L or K409E, as detailed above).

[0117] Thus, the first CH3-containing polypeptide described herein may comprise a K360D amino acid, optionally in addition to an amino acid mutation at position S364 (e.g., S364V, S364I, S364T, S364Q or S364L, as detailed above), and / or optionally in addition to an amino acid mutation at position K409 (e.g., K409I, K409L or K409E, as detailed above).

[0118] In one example, the heterodimeric protein comprises a first human IgG CH3-containing polypeptide and a second human IgG CH3-containing polypeptide, wherein the first human IgG CH3-containing polypeptide comprises further amino acid mutations at positions S364, K409 and / or K360. Optionally, said first human IgG CH3-containing polypeptide further comprises amino acid mutations L351D and L368E and said second human IgG CH3-containing polypeptide comprises amino acid mutations T366K and L351K.

[0119] For example, a first human IgG CH3-containing polypeptide described herein may comprise the S364V amino acid.

[0120] Alternatively, the first human IgG CH3-containing polypeptide described herein may comprise the S364I amino acid.

[0121] In another example, the first human IgG CH3-containing polypeptide described herein may comprise a S364T amino acid.

[0122] In a further example, the first human IgG CH3-containing polypeptide described herein may comprise the S364Q amino acid.

[0123] Alternatively, the first human IgG CH3-containing polypeptide described herein may comprise the S364L amino acid.

[0124] The first human IgG CH3-containing polypeptide described herein may comprise an amino acid mutation at position K409 instead of or in addition to an amino acid mutation at position S364 (e.g., S364V, S364I, S364T, S364Q or S364L, as detailed above).

[0125] Thus, the first human IgG CH3-containing polypeptide described herein may optionally comprise an amino acid mutation at position S364 (e.g., S364V, S364I, S364T, S364Q or S364L, as detailed above), as well as a K409I amino acid.

[0126] Alternatively, the first human IgG CH3-containing polypeptide described herein may optionally comprise a K409L amino acid in addition to an amino acid mutation at position S364 (e.g., S364V, S364I, S364T, S364Q or S364L, as detailed above).

[0127] In a further example, the first human IgG CH3-containing polypeptide described herein may optionally comprise a K409E amino acid in addition to an amino acid variant at position S364 (e.g., S364V, S364I, S364T, S364Q or S364L, as detailed above).

[0128] The first human IgG CH3-containing polypeptide described herein may comprise an amino acid mutation at position K360 instead of or in addition to an amino acid mutation at position S364 (e.g., S364V, S364I, S364T, S364Q or S364L, as detailed above) and / or a mutation at position K409 (e.g., K409I, K409L or K409E, as detailed above).

[0129] Thus, a first human IgG CH3-containing polypeptide described herein may optionally comprise a K360D amino acid, in addition to an amino acid mutation at position S364 (e.g. S364V, S364I, S364T, S364Q or S364L, as detailed above) and / or an amino acid mutation at position K409 (e.g. K409I, K409L or K409E, as detailed above).

[0130] In one example, a heterodimeric protein is described comprising a first human IgG CH3-containing polypeptide and a second human IgG CH3-containing polypeptide, wherein said first human IgG CH3-containing polypeptide comprises amino acid mutations L351D and L368E and said second human IgG CH3-containing polypeptide comprises amino acid mutations T366K and L351K, and wherein the first human IgG CH3-containing polypeptide comprises further amino acid mutations at positions S364, K409 and / or K360.

[0131] Thus, in one aspect there is provided a heterodimeric protein comprising a first human IgG CH3-containing polypeptide and a second human IgG CH3-containing polypeptide, wherein said first human IgG CH3-containing polypeptide comprises the amino acid mutations L351D and L368E, said second human IgG CH3-containing polypeptide comprises the amino acid mutations T366K and L351K, and said first human IgG CH3-containing polypeptide comprises a further amino acid mutation at position S364.

[0132] In particular examples, the amino acid at position 364 is a valine, isoleucine, threonine, glutamine, or leucine.

[0133] In one embodiment, the amino acid at position 364 is a valine.

[0134] In one embodiment, the amino acid at position 364 is an isoleucine.

[0135] In one embodiment, the amino acid at position 364 is a threonine.

[0136] In one embodiment, the amino acid at position 364 is a glutamine.

[0137] In one embodiment, the amino acid at position 364 is a leucine.

[0138] In another embodiment, there is provided a heterodimeric protein comprising a first human IgG CH3-containing polypeptide and a second human IgG CH3-containing polypeptide, said first human IgG CH3-containing polypeptide comprising the amino acid mutations L351D and L368E, said second human IgG CH3-containing polypeptide comprising the amino acid mutations T366K and L351K, and said first human IgG CH3-containing polypeptide comprising a further amino acid mutation at position K409.

[0139] In particular examples, the amino acid at position 409 is isoleucine, leucine, or glutamic acid.

[0140] In one embodiment, the amino acid at position 409 is an isoleucine.

[0141] In one embodiment, the amino acid at position 409 is a leucine.

[0142] In one embodiment, the amino acid at position 409 is glutamic acid.

[0143] In another embodiment, there is provided a heterodimeric protein comprising a first human IgG CH3-containing polypeptide and a second human IgG CH3-containing polypeptide, wherein said first human IgG CH3-containing polypeptide comprises the amino acid mutations L351D and L368E, said second human IgG CH3-containing polypeptide comprises the amino acid mutations T366K and L351K, and said first human IgG CH3-containing polypeptide comprises a further amino acid mutation at position K360.

[0144] In a particular example, the amino acid at position 360 is an aspartic acid.

[0145] The amino acid mutations described herein may refer to a single mutation at position S364, at position K409, or at position K360 in the "DE" arm of the heterodimeric protein of the invention. In a further embodiment of the invention, the heterodimeric protein has at least two mutations at a position selected from S364, K409 or K360. In a further embodiment of the invention, the heterodimeric protein has at least three further mutations at positions S364, K409 and K360.

[0146] Thus, in one aspect there is provided a heterodimeric protein comprising a first human IgG CH3-containing polypeptide and a second human IgG CH3-containing polypeptide, said first human IgG CH3-containing polypeptide comprising the amino acid mutations L351D and L368E, said second human IgG CH3-containing polypeptide comprising the amino acid mutations T366K and L351K, and said first human IgG CH3-containing polypeptide comprising further amino acid mutations at positions S364 and K409, wherein the amino acid at position 364 is valine, isoleucine, threonine, glutamine or leucine, and the amino acid at position 409 is isoleucine, leucine or glutamic acid.

[0147] For example, the first human IgG CH3-containing polypeptide described herein may comprise the amino acid S364V and the amino acid K409I. Alternatively, the first human IgG CH3-containing polypeptide described herein may comprise the amino acid S364V and the amino acid K409L. In one example, the first human IgG CH3-containing polypeptide described herein may comprise the amino acid S364V and the amino acid K409E.

[0148] In another example, the first human IgG CH3-containing polypeptide described herein may comprise the amino acid S364I and the amino acid K409I. Alternatively, the first human IgG CH3-containing polypeptide described herein may comprise the amino acid S364I and the amino acid K409L. In one example, the first human IgG CH3-containing polypeptide described herein may comprise the amino acid S364I and the amino acid K409E.

[0149] In another example, the first human IgG CH3-containing polypeptide described herein may comprise the amino acid S364T and K409I. Alternatively, the first human IgG CH3-containing polypeptide described herein may comprise the amino acid S364T and K409L. In one example, the first human IgG CH3-containing polypeptide described herein may comprise the amino acid S364T and K409E.

[0150] In another example, the first human IgG CH3-containing polypeptide described herein may comprise the amino acid S364Q and the amino acid K409I. Alternatively, the first human IgG CH3-containing polypeptide described herein may comprise the amino acid S364Q and the amino acid K409L. In one example, the first human IgG CH3-containing polypeptide described herein may comprise the amino acid S364Q and the amino acid K409E.

[0151] In another example, the first human IgG CH3-containing polypeptide described herein may comprise the amino acid S364L and the amino acid K409I. Alternatively, the first human IgG CH3-containing polypeptide described herein may comprise the amino acid S364L and the amino acid K409L. In one example, the first human IgG CH3-containing polypeptide described herein may comprise the amino acid S364L and the amino acid K409E.

[0152] In another embodiment, there is provided a heterodimeric protein comprising a first human IgG CH3-containing polypeptide and a second human IgG CH3-containing polypeptide, wherein said first human IgG CH3-containing polypeptide comprises the amino acid mutations L351D and L368E and said second human IgG CH3-containing polypeptide comprises the amino acid mutations T366K and L351K, and wherein the first human IgG CH3-containing polypeptide comprises further amino acid mutations at positions S364 and K360, wherein the amino acid at position 364 is valine, isoleucine, threonine, glutamine or leucine and the amino acid at position 360 is aspartic acid.

[0153] For example, a first human IgG CH3-containing polypeptide described herein may comprise the amino acids S364V and K360D.

[0154] In another example, a first human IgG CH3-containing polypeptide described herein may comprise the amino acids S364I and K360D.

[0155] In another example, a first human IgG CH3-containing polypeptide described herein may comprise the amino acids S364T and K360D.

[0156] In another example, a first human IgG CH3-containing polypeptide described herein may comprise the amino acids S364Q and K360D.

[0157] In another example, a first human IgG CH3-containing polypeptide described herein may comprise the amino acids S364L and K360D.

[0158] In another aspect, there is provided a heterodimeric protein comprising a first human IgG CH3-containing polypeptide and a second human IgG CH3-containing polypeptide, said first human IgG CH3-containing polypeptide comprising the amino acid mutations L351D and L368E and said second human IgG CH3-containing polypeptide comprising the amino acid mutations T366K and L351K, wherein the first human IgG CH3-containing polypeptide comprises further amino acid mutations at positions K409 and K360, wherein the amino acid at position 409 is isoleucine, leucine or glutamic acid and the amino acid at position 360 is aspartic acid.

[0159] For example, a first human IgG CH3-containing polypeptide described herein may comprise the amino acids K409I and K360D.

[0160] In another example, a first human IgG CH3-containing polypeptide described herein may comprise the amino acids K409L and K360D.

[0161] In another example, a first human IgG CH3-containing polypeptide described herein may comprise the amino acids K409E and K360D.

[0162] In another aspect, there is provided a heterodimeric protein comprising a first human IgG CH3-containing polypeptide and a second human IgG CH3-containing polypeptide, said first human IgG CH3-containing polypeptide comprising the amino acid mutations L351D and L368E and said second human IgG CH3-containing polypeptide comprising the amino acid mutations T366K and L351K, wherein the first human IgG CH3-containing polypeptide comprises further amino acid mutations at positions S364, K409 and K360, wherein the amino acid at position 364 is valine, isoleucine, threonine, glutamine or leucine, the amino acid at position 409 is isoleucine, leucine or glutamic acid, and the amino acid at position 360 is aspartic acid.

[0163] For example, the first human IgG CH3-containing polypeptide described herein may include S364V, K409I and K360D. Alternatively, the first human IgG CH3-containing polypeptide described herein may include S364V, K409L and K360D. In one example, the first human IgG CH3-containing polypeptide described herein may include S364V, K409E and K360D.

[0164] In another example, the first human IgG CH3-containing polypeptide described herein may include S364I, K409I, and K360D. Alternatively, the first human IgG CH3-containing polypeptide described herein may include S364I, K409L, and K360D. In one example, the first human IgG CH3-containing polypeptide described herein may include S364I, K409E, and K360D.

[0165] In another example, the first human IgG CH3-containing polypeptide described herein may include S364T, K409I, and K360D. Alternatively, the first human IgG CH3-containing polypeptide described herein may include S364T, K409L, and K360D. In one example, the first human IgG CH3-containing polypeptide described herein may include S364T, K409E, and K360D.

[0166] In another example, the first human IgG CH3-containing polypeptide described herein may include S364Q, K409I, and K360D. Alternatively, the first human IgG CH3-containing polypeptide described herein may include S364Q, K409L, and K360D. In one example, the first human IgG CH3-containing polypeptide described herein may include S364Q, K409E, and K360D.

[0167] In another example, the first human IgG CH3-containing polypeptide described herein may include S364L, K409I, and K360D. Alternatively, the first human IgG CH3-containing polypeptide described herein may include S364L, K409L, and K360D. In one example, the first human IgG CH3-containing polypeptide described herein may include S364L, K409E, and K360D.

[0168] In a particular embodiment, the amino acid at position 360 is an aspartic acid and the amino acid at position 364 is an isoleucine.

[0169] In another embodiment, the amino acid at position 360 is an aspartic acid and the amino acid at position 364 is a threonine.

[0170] In a further embodiment, the amino acid at position 360 is an aspartic acid and the amino acid at position 364 is a valine.

[0171] In yet another embodiment, the amino acid at position 360 is an aspartic acid and the amino acid at position 409 is a glutamic acid.

[0172] In a further embodiment, the amino acid at position 364 is a leucine and the amino acid at position 409 is a leucine.

[0173] In yet another embodiment, the amino acid at position 364 is a threonine and the amino acid at position 409 is a glutamic acid.

[0174] Mutant polypeptides can be made by any suitable means known in the art. The variants described herein may be based on the human IgG CH3 domain, but one or more of the parent amino acids (positions 360, 364 and / or 409) are mutated at the corresponding positions of the human IgG CH3 amino acids described herein.

[0175] The sequence of the human IgG CH3 domain is well known in the art. By changing one or more amino acids of the polypeptide, the side chains at those positions are altered. The amino acid mutations described herein can be introduced into the nucleic acid encoding the CH3 domain or into the CH3 domain itself by various means, including genetic means using molecular biology, or by enzymatic or chemical means. Suitable methods for generating the mutants described herein are well known in the art. For example, mutant nucleic acids can be generated de novo through nucleic acid synthesis encoding the mutant sequence and ordered through a number of providers. Similarly, mutant nucleic acids can be generated by mutagenesis techniques well known in the art. Such nucleic acids encoding mutant polypeptides can be cloned into host cells, expressed, and assayed as needed. These can be performed using well-known procedures, and various methods that can be used are described in Molecular Cloning - A Laboratory Manual, 3rd Ed. (Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001), and Current Protocols in Molecular Biology (John Wiley & Sons), the contents of which are incorporated by reference in their entirety. The nucleic acid encoding the variant can be incorporated into an expression vector to express the protein. The expression vector generally includes a promoter linked to control or regulatory sequences, a selectable marker, any fusion partners and / or additional elements. The variant can be produced by culturing a host cell transformed with a nucleic acid, preferably an expression vector, containing the variant-encoding nucleic acid under appropriate conditions that induce or cause expression of the protein. A wide variety of suitable host cells can be used, including, but not limited to, mammalian cells, bacteria, insect cells and yeast. For example, various cell lines that find use are described in the ATCC cell line catalog, available from the American Type Culture Collection, the contents of which are incorporated by reference in their entirety.Methods of introducing exogenous nucleic acid into host cells are well known in the art and vary depending on the host cell used.

[0176] In one aspect of the present invention, a method according to the present invention is provided for producing at least two Ig-like molecules or for producing heterodimeric Ig-like molecules, in which each of the CH3 domain-containing polypeptide chains further comprises a variable region that recognizes a different target epitope, and the target epitopes are located on the same molecule. This often allows for a more efficient counteraction of the (biological) function of said target molecule compared to the situation where only one epitope is targeted. For example, a heterodimeric immunoglobulin-like molecule can simultaneously bind to two epitopes present, for example, on a growth factor receptor or a soluble molecule important for the proliferation of tumor cells, thereby effectively blocking several independent signal transduction pathways that lead to uncontrolled proliferation, and at least two Ig-like molecules can simultaneously bind to two, or even three or four epitopes present on such growth factor receptors or soluble molecules.

[0177] In one embodiment, the target molecule is a soluble molecule, hi another embodiment, the target molecule is a membrane-bound molecule.

[0178] In another aspect of the present invention, the method of the present invention is provided for making one or more heterodimeric immunoglobulin-like molecules, each of the CH3 domain-containing polypeptide chains further comprising a variable region that recognizes a target epitope, and the target epitopes are located on different molecules. In this case, each of the different target molecules can be either a soluble molecule or a membrane-bound molecule. In one embodiment, the different target molecules are soluble molecules. Alternatively, one target molecule is a soluble molecule and the other target molecule is a membrane-bound molecule. In yet another embodiment, both target molecules are membrane-bound molecules. In one embodiment, the different target molecules are expressed in the same cell, while in other embodiments, the different target molecules are expressed in different cells. As a non-limiting example, any heterodimeric immunoglobulin-like molecule or any combination of at least two immunoglobulin-like molecules may be suitable for simultaneously blocking multiple membrane-bound receptors, neutralizing multiple soluble molecules such as cytokines or tumor cell growth factors, or neutralizing different virus serotypes or virus strains.

[0179] One embodiment provides a method according to the invention for producing one or more heterodimeric immunoglobulin-like molecules, where at least one of said target epitopes is located on a tumor cell. Alternatively or additionally, at least one of said target epitopes is located on the surface of an effector cell. This is suitable for example for recruiting T cells or NK cells to kill tumor cells. For example, at least one Ig-like molecule is produced by the method according to the invention, which can recruit immune effector cells, preferably human immune effector cells, by specifically binding to a target molecule located on the immune effector cell. In a further embodiment, said immune effector cells are activated when the immunoglobulin-like molecule binds to the target molecule. Recruitment of effector mechanisms can include redirection of immune-modulating cytotoxicity, for example by administering an immunoglobulin-like molecule produced by the method according to the invention, which can bind to a cytotoxicity-inducing molecule, such as a T cell receptor or an Fcγ receptor, thereby activating downstream immune effector pathways. The term "immune effector cell" or "effector cell" as used herein refers to a cell within the natural repertoire of cells of the mammalian immune system that can be activated to affect the survival rate of a target cell. Immune effector cells include cells of the lymphoid lineage, such as natural killer (NK) cells, T cells, including cytotoxic T cells, or B cells, but also cells of the myeloid lineage, such as monocytes or macrophages, dendritic cells, and neutrophil granulocytes. Thus, said effector cells are preferably NK cells, T cells, B cells, monocytes, macrophages, dendritic cells, or neutrophil granulocytes.

[0180] Thus, in one aspect, the present invention provides a method according to the present invention for producing a heterodimeric immunoglobulin-like polypeptide, in which each of the CH3 domain-containing polypeptide chains further comprises a variable region that recognizes a target epitope. In one embodiment, each of the two variable regions of the CH3 domain-containing polypeptide chains recognizes the same target epitope, but with different affinities. In another embodiment, each of the two variable regions of the CH3 domain-containing polypeptide chains recognizes a different target epitope. In another embodiment, the different target epitopes are located on the same target molecule, which may be either a membrane-bound or soluble molecule. In another embodiment, the different target epitopes are located on different target molecules, which may be found on the same cell or different cells. Alternatively, the different target molecules may be soluble molecules, or one target molecule may be a soluble molecule and the other target molecule may be a membrane-bound molecule. In one embodiment, at least one of the target molecules of the heterodimeric immunoglobulin-like polypeptide is located on a tumor cell. In yet another embodiment, at least one of the target molecules of the heterodimeric Ig-like polypeptide is located on an effector cell.

[0181] In one embodiment, there is provided a method according to the invention for producing one or more heterodimeric immunoglobulin-like polypeptides, said polypeptides being antibodies. In one embodiment, the antibodies are IgG, IgA, IgD, IgE or IgM isotype antibodies. In one embodiment, the antibodies are IgG isotype antibodies. In another embodiment, the antibodies are IgG1 isotype antibodies.

[0182] Further provided is a heterodimeric immunoglobulin-like polypeptide or a mixture of at least two Ig-like polypeptides obtainable by the method according to the invention, said heterodimeric polypeptide or mixture of polypeptides comprising two CH3 domains, where one of said two CH3 domains comprises the amino acid mutations L351D and L368E and the other of said two CH3 domains comprises the amino acid mutations T366K and L351K, which comprise one or more mutations relative to the wild-type CH3 sequence that enhance heterodimerization and / or destabilize homodimer formation, preferably comprising mutations at positions 360, 364 and 409 according to EU numbering that achieve a combination of these advantages.

[0183] These amino acid mutations cause preferential pairing of the two CH3 domains as described above. The amino acid mutations L351D and L368E in one of the two CH3 domains and the amino acid mutations T366K and L351K in the other of the two CH3 domains are collectively referred to herein as DEKK. The CH3 domain with the amino acid mutations L351D and L368E is also referred to as "DE side chain", "DE arm" or "DE", and the CH3 domain with the amino acid mutations T366K and L351K is also referred to as "KK side chain", "KK arm" or "KK". The amino acid mutations described herein (positions K360, S364 and / or K409) can be introduced alone or preferably together with other mutations to promote heterodimerization, more preferably on the DEKK backbone, more preferably on the DE side chain. On the other hand, the mutations at amino acid positions 360, 364 and 409 can enhance the stability of the heterodimer and / or destabilize the homodimer.

[0184] Also provided is a pharmaceutical composition comprising a heterodimeric immunoglobulin-like polypeptide or a mixture of at least two polypeptides obtainable by any method according to the invention. In one embodiment, the heterodimeric polypeptide is an antibody(ies) or a multivalent multimer. The pharmaceutical composition may comprise a heterodimeric immunoglobulin-like polypeptide or a mixture of at least two polypeptides comprising a monospecific or bispecific Ig-like molecule, or a combination of monospecific and bispecific Ig-like polypeptides. Additionally, the pharmaceutical composition according to the invention comprises a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes physiologically compatible solvents, salts, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Depending on the route of administration (e.g., intravenous, subcutaneous, intraarticular, etc.), the Ig-like polypeptide may be coated with a material that protects the Ig-like polypeptide from the action of acids and other natural conditions that may inactivate the Ig-like polypeptide.

[0185] In one embodiment, the present invention provides a heterodimeric antibody comprising a first heavy chain having a first human IgG CH3 region and a second heavy chain having a second human IgG CH3 region different from the first human IgG CH3 region, wherein the first human IgG CH3 region comprises IgG CH3 amino acid mutations L351D and L368E, and the second human CH3 region comprises human IgG CH3 amino acid mutations T366K and L351K, and wherein the first human IgG CH3 region comprises an additional amino acid mutation at position K360, S364 or K409. The heterodimeric antibody preferably comprises one or more antibody light chains. The first and second human IgG CH3 regions are preferably human IgG1, IgG2, IgG3 or IgG4 regions. The first and second human IgG CH3 regions are preferably IgG1 CH3 regions. The first heavy chain may comprise a heavy chain variable region different from the heavy chain variable region of the second heavy chain. Antibody variable domains comprising different heavy chain variable regions associated with appropriate light chains can bind to different epitopes and / or antigens. The first heavy chain and / or the second heavy chain can comprise two or more variable regions. Heterodimeric antibodies comprising one or two heavy chains with two or more variable regions can form three or more variable domains with appropriate complementary variable regions. Heterodimeric antibodies with two or more variable domains that bind to different epitopes and / or antigens are also referred to as bispecific antibodies, trispecific antibodies, tetraspecific antibodies, etc. Such antibodies are also collectively referred to as multispecific antibodies.

[0186] Although for clarity and conciseness of description, features are described herein as part of the same or separate embodiments, it will be understood that the scope of the invention may include embodiments having all or a combination of some of the described features.

[0187] The present invention is further illustrated by the following examples, which in no way limit the invention but merely serve to clearly describe it.

[0188] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. Also, as used herein, the singular terms "a" and "an" include the plural meaning. Unless otherwise specified, nucleic acids are written from left to right in 5' to 3' orientation, and amino acid sequences are written from left to right in amino to carboxy orientation, respectively. It is understood that the present invention is not limited to the specific methodology, protocols, and reagents described, which may vary depending on the context in which they are used by those skilled in the art.

[0189] Details of the invention are supported by the following non-limiting examples. EXAMPLES

[0190] Working Example Example 1: In silico prediction A human IgG Fc mutant containing DEKK (L351D, L368E / L'351K, T'366 K) was used as a starting model. WO2013 / 157954; De Nardis et al, J. Biol. Chem. (2017) 292(35) 14706-14717. In silico structural studies were performed to identify key contact residues in the CH3 DEKK / DEDE interface. These residues were analyzed using High Ambiguity Driven protein-protein DOCKing (HADDOCK; an information-driven docking approach that models the docking of biomolecular complexes such as proteins or nucleic acids) to identify mutations that significantly reduce the formation of DEDE homodimers, but not significantly negatively affect or improve the stability of the DEKK interface. A total of 18 human IgG CH3 mutants were selected for in vitro analysis, all in the DEKK backbone. Each of the 18 mutants had a different amino acid mutation at one of the following positions within the human IgG DE CH3 domain: K360, S354, S364 or K409.

[0191] Example 2: In vitro SDS-PAGE study We produced an IgG vector containing all the proposed mutations in CH3 in addition to the L351D, L368E (DE) mutations. For this purpose, 18 constructs were generated. Production was performed with transfections of a single DE* heavy chain, and a combination of DE* and KK heavy chains (where * denotes one of the 18 mutants mentioned above). Constructs were cloned into MG1122C1708 (Figure 1) using BspEI and AflII restriction enzymes. To obtain all mutants, colony / sequencing PCR was performed on the ligation products. All confirmed mutants were miniprepped and then sequenced to confirm the identity of CH2, CH3 and VH. Transfections into 293FF cells of single DE* constructs (labeled PGxxxx IgG) and DE* constructs combined with KK constructs with different VHs (labeled PBxxxxx bispecific IgG) were performed. After 7 days of incubation (37°C, 8% CO2, 155 rpm), cell supernatants were harvested and subjected to Protein A purification (acidic elution) and buffer exchange into PBS pH 7.4, and the IgG concentration of the resulting samples was measured by Octet.

[0192] SDS-PAGE analysis All produced and purified IgG was analyzed by non-reducing SDS-PAGE. For each production plate, the same amount of IgG sample was added to each well, usually 1 μg (if this was not possible, the amount was adjusted to the lowest concentration sample). This allowed direct comparisons to be made between samples. Gels were visually inspected for their intact IgG and half-body content. The relative amounts of intact IgG and half-bodies in each gel could also be quantified using Chemidoc MP Imager software. The high amount of half-bodies in single (PGxxxx) transfections indicated that the variant homodimers were unstable. The IgG bands in double transfections (PBxxxxx) confirmed that heterodimers were formed.

[0193] DE*-CH3 mutants that mostly showed half-body formation in single transfections (comparable to the MG4539C1452 control sample containing the KK CH3 region) and mostly formed full IgG in bispecific transfections were selected for further characterization in follow-up studies (Figures 2-4 and Table 1). A total of three of the four positions identified in silico showed improved properties in vitro compared to the control sample (K360, S364 and K409). A total of nine mutants were selected for further studies.

[0194] [Table 1]

[0195] Following the SDS-PAGE experiments, further in silico HADDOCK analysis was performed to identify potentially promising double mutants (Table 2, rows 10–15) that could be further improved compared to the control. [Table 2]

[0196] Taken together, a total of 18 mutants were created at three positions in the DE arm of CH3. Single transfections were performed to obtain homodimers and double transfections with a common KK arm to obtain bispecific IgG. SDS-PAGE analysis showed that five DEKK mutants showed more half-body contamination in the single arm product than in the DEKK control sample. Due to the bispecific IgG results in the double transfection studies, three further mutants were selected despite the aberrant SDS-PAGE results being single production. Using HADDOCK, the high-performing mutation combinations were analyzed, from which six further combinations were selected for further analysis.

[0197] Example 3: In vitro stability results and biophysical properties The 15 selected mutants were produced as bispecifics (CD137 (MF6744 - previously published in U.S. Publication No. 2020 / 0017595A1; U.S. Publication No. 2019 / 00352401A2) x fibrinogen (MF1122)) containing DE:KK ratios of 1:1 and 3:1, where the DE arm was overexpressed to test its homodimer formation.

[0198] The IgGs were further characterized using CIEX-HPLC (cation exchange high performance liquid chromatography), HP-SEC (high performance size exclusion chromatography) and nMS (native mass spectrometry) techniques. In addition, a stability analysis was performed analyzing the melting and aggregation temperatures Tm and Tagg. Mutants that significantly reduced homodimer formation and showed good Fc stability as indicated by high Tm / Tagg were selected for larger scale protein production.

[0199] The VHs used have different weights (monoclonal IgG MF1122 has a weight of 144905 kDa, MF6744 has a weight of 145932 kDa), therefore homodimers and heterodimers can be separated by nMS. As they have different CIEX RTs (13.3 and 9.7 min), especially as they bind KK and DE, respectively, homodimers and heterodimers can be separated by CIEX. The Fab has a high Tm. The high Tm Fab allowed us to identify the melting temperature (Tm) of the mutant CH3.

[0200] The resulting IgG with a 1:1 DNA ratio was analyzed by Tm / Tagg analysis using an UNcle instrument (UNchained Labs product code 200-1037). The melting temperature (Tm) is determined by following the change in the intrinsic fluorescence of the amino acids upon increasing temperature (spectral range 250-720 nM). The aggregation temperature (Tagg) is determined by measuring static light scattering (SLS) simultaneously with the measurement of Tm, using lasers with wavelengths of 266 nm and 473 nm, detecting the scattered light at an angle of 90°. DLS (Dynamic Light Scattering) analysis used a temperature gradient from 25 °C to 95 °C at a rate of 0.3 °C / min for the determination of Tm / Tagg by fluorescence and SLS acquisition (Blue&UV, exposure time 1000 ms, UV266: filter 4, Blue473: filter 3).

[0201] This allowed the determination of Tm / Tagg for unstressed samples with a KK:DE ratio of 1:1. As shown in Table 4, all samples were at 300 μg / mL.

[0202] Stress testing Small scale stability assays were performed by stressing the modified IgG, all of which were performed in PBS. Three 100 μL aliquots of each sample were generated at 300 μg / mL (see Table 3).

[0203] The following stresses were applied to the samples: - 5xFT (freeze-thaw) (samples were repeatedly stored at -80°C for at least 1 h and then completely thawed at room temperature); - 50°C for 2 days (place samples in a 50°C water bath or incubator and incubate for 2 days); Store at 4℃ (as a non-stressed control).

[0204] The stressed samples were then analyzed by DLS and directly compared to unstressed samples (held at 4°C) to confirm the presence of aggregates in the samples.

[0205] Although some differences in the Tm / Tagg of the variants were observed, they were all considered stable molecules (Tables 3 and 4). [Table 3]

[0206] [Table 4]

[0207] HP-SEC The produced IgG was analyzed by HP-SEC according to standard methods known in the art: Agilent 1260 series HPLC system, Tosoh TSK-gel G3000SWxl Cat. No. 808541, Tosoh TSK guard column SWXL Cat. No. 808543, running buffer: 200 mM NaPO4, 50 mM NaCl, pH 7.0, 20 μg sample added. HP-SEC analysis was performed to detect small aggregates compared to DLS analysis (Table 5) and was performed after 1x F / T cycle to represent samples typically used in the study after storage at -80°C.

[0208] [Table 5]

[0209] Stress tests show that the DEKK mutant is highly stable, exhibiting aggregation only after severe stress. [Table 6]

[0210] [Table 7]

[0211] [Table 8]

[0212] CIEX-HPLC The produced IgG was analyzed by CIEX-HPLC using an Agilent 1260 series HPLC system, Tosoh TSKgel SP-STAT column, catalog number 21964, buffers used: A) 25 mM NaPO4, pH 6.0 and B) 25 mM NaPO4, 1 M NaCl, pH , analyzed by a linear gradient of 0-30% buffer B (2% B increase per mL), 0.5 mL / min, adding 10 µg of sample. The profiles generated were analyzed for peak shape, retention time, bispecific / contaminant ratio, and separation of bispecific and contaminant materials (most pronounced in the 3:1 transfected samples). The separation between wt PG1122 and wt PG6744 is approximately 3.7 min and is increased by KK / DE and other mutants. The WT DEKK control (ctrl.) showed peaks at 10.25 min (biclonic) and 7.8 min (DEDE homodimer), as expected. Retention time (RT) values ​​are nearly identical for products with either a 1:1 or 1:3 ratio.

[0213] In some samples, early eluting species consisting of DEDE homodimers were seen in one or both products, whereas other samples had no detectable early eluting species.

[0214] Most notably, large amounts of early eluting species were observed in the S354Q mutant (both products), the S364T mutant and the WT (3:1 production), but were less than 10% in all other samples.

[0215] All samples showed a pre-peak. A larger pre-peak was observed in the 1:3 production, approximately 3-fold larger than that observed in the 1:1 production, relative to the height of the main peak. Regardless of theory, we believe that the DE* half-body species did not bind to the column and eluted in the "pre-peak" (dead volume of the column) due to the additional negative charges present in certain variants.

[0216] Many of the mutants were highly successful in preventing homodimer formation, as evidenced by the absence of early eluting peaks.

[0217] Taken together, for 15 out of 18 IgGs generated, very little or no DEDE homodimer was formed by CIEX-HPLC, even at 3:1 overexpression of the (modified) DE arm, indicating that most of the additional mutants were successful in preventing or reducing homodimer formation compared to the DEKK control (Table 9).

[0218] [Table 9]

[0219] LabCHIP To determine the amount of IgG (DEKK+DEDE) and half-bodies in the Biclonic product samples, LabChips were used. Samples were analyzed at a concentration of 0.3 μg / μL using a Perkin Elmer LabCihp GXII Touch HT, Protein Clear HR Reagent kit (Cat. No. CLS960014) and Protein Express Assay LabChip (Cat. No. 760499) according to the manufacturer's instructions. It was observed that the formation of DE half-bodies differed significantly between the variants, and as expected, the 3:1 production contained more DE half-bodies (Table 10).

[0220] [Table 10]

[0221] nMS For all generated IgG, 100 μg / sample was analyzed by nMS analysis (see methodology in Rosati et al, Anal Chem. 2012 Aug 21;84(16):7227-32). The relative proportions between the different species (bispecifics, homodimers, half-bodies) were investigated.

[0222] All 16 samples were successfully analyzed and the IgG species could be identified by this technique. In the control DEDE sample, 95% bispecificity was obtained in 1:1 production. The percentage of DEKK mutant heterodimer formation in 1:1 production varied from 74% to 100%. A total of 12 samples contained 90% or more DEKK heterodimers. Homodimer formation was significantly reduced in 14 samples compared to the DEKK control. In eight samples, 2% or less DEDE homodimers were formed in 3:1 production.

[0223] Taken together, 14 mutants significantly reduced homodimer formation at 3:1 production (compared to the DEKK control), and eight mutants barely formed homodimers at all (Table 11).

[0224] [Table 11]

[0225] Summary of results Different methods were used for the detection of half-bodies, including LabChip, HP-SEC and nMS. Although the values ​​obtained differed in absolute terms, with nMS detecting fewer half-bodies than the other methods, the final conclusions regarding the utility of the mutants in reducing DEDE homodimer formation were consistent (Table 12). Different techniques were used to characterize the samples for the presence of DEKK, IgG-derived contaminants and aggregates, and although the values ​​differed strictly, consistent conclusions could be drawn from the data obtained (Tables 13-15).

[0226] [Table 12]

[0227] [Table 13]

[0228] [Table 14]

[0229] [Table 15]

[0230] Taken together, the variants provided herein increase heterodimer stability and decrease homodimer formation based on changes at the CH3 interface of the heterodimeric polypeptide.

[0231] Example 4: The effect of the mutants shown in Table 16 on DEKK heterodimer formation with respect to yield, purity and formation of DE contaminants was further investigated. In this particular example, bispecific proteins with mutations in the DE heavy chain were produced with a KK-containing heavy chain at various DE:KK ratios as described above, purified using Protein A, analyzed by CIEX and analyzed by LabChip / SDS-PAGE.

[0232] Cloning: The VH sequences of anti-TT on the DE side and anti-CD3 on the KK side were cloned with SfiI and XhoI into a vector containing the following mutants: K360D_K409E (SEQ ID NO: 12), S364L_K409L (SEQ ID NO: 14), S364L (SEQ ID NO: 2) and S364T_K409E (SEQ ID NO: 13). Colony / sequencing PCR was performed on the ligation products to obtain all mutants. All confirmed mutants were miniprepped and then sequenced. IgG was produced by transfection of single DE* constructs (labeled PGxxxx IgG) and DE* constructs combined with KK constructs (labeled PBxxxxx bispecific IgG) at various DE:KK ratios (Table 16) into 293FF cells. After 7 days of incubation (37°C, 8% CO2, 155 rpm), cell supernatants were harvested and subjected to Protein A purification (acidic elution) and buffer exchange into PBS pH 7.4. The IgG concentration of the samples was measured, and the proteins were then characterized by HP-SEC, CIEX and Lab-Chip / SDS-PAGE.

[0233] [Table 16]

[0234] HP-SEC: The single-arm products were analyzed by HP-SEC according to the protocol described in Example 3. The same amount of each IgG was used, but not less than 10 µg. For all variants, the DE arms perform under native conditions as more than 75% half-bodies, about 10-20% homodimers, and some aggregates (Table 17).

[0235] [Table 17]

[0236] CIEX analysis: IgG proteins were analyzed by CIEX (protocol described in Example 3). As a control, the single-arm products were analyzed to be able to assign the peaks detected in the bispecific samples. The amount of protein injected was adjusted in all samples. The protein amount of the samples containing a single arm was adjusted to 5 μg in each sample. If the concentration was too low, the maximum possible amount (100 μL) was injected. The expected retention time (RT) of the WT-DE homodimer with anti-TT heavy chain (VH; SEQ ID NO: 16) is 13 min. The RT of the KK half-body with anti-CD3 heavy chain (VH; SEQ ID NO: 17) is 26 min. The retention time of the KK-KK dimer elutes too late to be detected. The quantification of the components in the samples did not take into account pre-peaks. Peaks belonging to the same species (PB, DE or KK) were summed and added as one value. The composition of the sample eluting from the CIEX column is based on the percentage of the area of ​​each peak.

[0237] Heterodimer purity was improved for all mutants examined under the conditions tested when the same ratios were compared (Table 18). The effect of altering the DE:KK ratio was such that an increase in KK DNA, and therefore an increase in translation of the KK polypeptide, resulted in a decrease in the DE contaminant.

[0238] [Table 18]

[0239] In single-arm production, the mutants showed a decrease in DEDE homodimers and an increase in DE half-bodies (Table 19), a result consistent with the data generated using HP-SEC.

[0240] [Table 19]

[0241] Effect of mutations on purity The amount of bispecific protein present in each sample was estimated using the CIEX results and discussed in terms of the purity of the bispecific protein. The purity of the heterodimeric PB protein is improved in this example with Protein A purification. Increasing the DE:KK ratio improves the purity (Table 20).

[0242] [Table 20]

[0243] Sequence used: >MV1708_DE_S364V [Sequence number 1] TCCGGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTTTCGAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCGACCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCGTCCTGACCTGCGAGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTTTAACGGATCTTAATTAATCCGAGCTCGGTACCAAGCTTAAG >MV1708_DE_S364L [SEQ ID NO: 2] TCCGGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTTTCGAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCGACCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCCTCCTGACCTGCGAGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTTTAACGGATCTTAATTAATCCGAGCTCGGTACCAAGCTTAAG >MV1708_DE_S364I [SEQ ID NO: 3] TCCGGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTTTCGAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCGACCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCATCCTGACCTGCGAGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTTTAACGGATCTTAATTAATCCGAGCTCGGTACCAAGCTTAAG >MV1708_DE_S364T [SEQ ID NO: 4] TCCGGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTTTCGAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCGACCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCACACTGACCTGCGAGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTTTAACGGATCTTAATTAATCCGAGCTCGGTACCAAGCTTAAG >MV1708_DE_S364Q [SEQ ID NO: 5] TCCGGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTTTCGAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCGACCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCCAGCTGACCTGCGAGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTTTAACGGATCTTAATTAATCCGAGCTCGGTACCAAGCTTAAG

[0244] >MV1708_DE_K409I [SEQ ID NO: 6] TCCGGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTTTCGAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCGACCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCGAGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCATCCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTTTAACGGATCTTAATTAATCCGAGCTCGGTACCAAGCTTAAG >MV1708_DE_K409L [SEQ ID NO: 7] TCCGGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTTTCGAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCGACCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCGAGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCCTGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTTTAACGGATCTTAATTAATCCGAGCTCGGTACCAAGCTTAAG >MV1708_DE_K409E [SEQ ID NO: 8] TCCGGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTTTCGAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCGACCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCGAGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCGAACTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTTTAACGGATCTTAATTAATCCGAGCTCGGTACCAAGCTTAAG >CH2-3_DE_K360D-S364I [SEQ ID NO: 9] Tccggaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtttcgaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccgaccccccatcccgggaggagatgaccgacaaccaggtcatcctgacctgcgaggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgagtttaacggatcttaattaatccgagctcggtaccaagcttaag >CH2-3_DE_K360D-S364T [SEQ ID NO: 10] Tccggaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtttcgaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccgaccccccatcccgggaggagatgaccaagaaccaggtcctcctgacctgcgaggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcctgctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgagtttaacggatcttaattaatccgagctcggtaccaagcttaag

[0245] >CH2-3_DE_K360D-S364V [SEQ ID NO: 11] Tccggaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtttcgaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccgaccccccatcccgggaggagatgaccaagaaccaggtcctcctgacctgcgaggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcctgctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgagtttaacggatcttaattaatccgagctcggtaccaagcttaag >CH2-3_DE_K360D-K409E [SEQ ID NO: 12] Ccggaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtttcgaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccgaccccccatcccgggaggagatgaccgacaaccaggtcagcctgacctgcgaggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgagtttaacggatcttaattaatccgagctcggtaccaagcttaag >CH2-3_DE_S364T-K409E [SEQ ID NO: 13] tccggaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtttcgaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccgaccccccatcccgggaggagatgaccaagaaccaggtcacactgacctgcgaggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgagtttaacggatcttaattaatccgagctcggtaccaagcttaag >CH2-3_DE_S364L-K409L [SEQ ID NO: 14] tccggaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtttcgaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccgaccccccatcccgggaggagatgaccaagaaccaggtcctcctgacctgcgaggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcctgctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgagtttaacggatcttaattaatccgagctcggtaccaagcttaag >MV1708_DE_K360D [SEQ ID NO: 15] TCCGGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTTTCGAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCGACCCCCCATCCCGGGAGGAGATGACCGACAACCAGGTCAGCCTGACCTGCGAGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTTTAACGGATCTTAATTAATCCGAGCTCGGTACCAAGCTTAAG >VH sequence of anti-TT Fab [SEQ ID NO: 16] EVQLVETGGGVVQPGRSLRLSCAASGFTFSQYAMHWVRQAPGKGLEWVAIISHDERNKYYVDSGMGRFTISRDNSKNTLFLQMNSLRSEDTAVYYCARDMRKGGYYYGFDVWGQGTTVTVSS >VH sequence of anti-CD3 Fab [SEQ ID NO: 17] QVQLVESGGGVVQPGRSLRLSCAASGFTFRSYGMHWVRQAPGKGLEWVAIIWYSGSKKNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGTGYNWFDPWGQGTLVTVSS

[0246] The reader's attention is directed to all documents and literature related to this application that have been filed contemporaneously or prior to this specification and that are open to public inspection together with this specification, the contents of which are incorporated herein by reference.

[0247] All features disclosed in this specification (including the accompanying claims, abstract and drawings), and / or all steps of any method or process so disclosed, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive.

[0248] Each feature disclosed in this specification (including the accompanying claims, abstract and drawings), unless expressly stated otherwise, may be replaced by an alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only one generic example of a series of equivalent or similar features.

[0249] The invention is not limited to the details of any of the above embodiments.The invention extends to any novel, or any novel combination of features disclosed in this specification (including the accompanying claims, abstract and drawings), or to any novel, or any novel combination of method or process steps so disclosed.

Claims

1. 1. An isolated heterodimeric protein comprising a first human IgG CH3-containing polypeptide and a second human IgG CH3-containing polypeptide, wherein said first human IgG CH3-containing polypeptide comprises amino acid mutations L351D and L368E, said second human IgG CH3-containing polypeptide comprises amino acid mutations T366K and L351K, and said first human IgG CH3-containing polypeptide further comprises amino acid mutations at positions S364, K409 and / or K360.

2. a) the amino acid at position 364 is valine, isoleucine, threonine, glutamine or leucine; and / or b) the amino acid at position 409 is isoleucine, leucine, or glutamic acid; and / or c) the amino acid at position 360 is aspartic acid; 2. The isolated heterodimeric protein of claim 1 .

3. 3. The isolated heterodimeric protein of claim 1 or 2, wherein the heterodimeric protein comprises a human IgG1, IgG2, IgG3 or IgG4 CH3 region.

4. 4. The isolated heterodimeric protein of claim 1, wherein the heterodimeric protein comprises a human immunoglobulin Fc region, optionally wherein the human immunoglobulin Fc region comprises an IgG1, IgG2, IgG3 or IgG4 Fc region.

5. a) the first CH3-containing polypeptide is an antibody heavy chain; and / or b) the second CH3-containing polypeptide is an antibody heavy chain; and / or c) the heterodimeric protein further comprises one or more antibody light chains; and / or d) the antibody light chain is a conventional antibody light chain; 5. An isolated heterodimeric protein according to any one of claims 1 to 4.

6. A pharmaceutical composition comprising the isolated heterodimeric protein of any one of claims 1 to 5.

7. 6. An isolated nucleic acid encoding the first and second human IgG CH3-containing polypeptides of any one of claims 1 to 5.

8. A recombinant host cell comprising the isolated nucleic acid of claim 7.

9. 10. A method for producing an isolated heterodimeric protein according to any one of claims 1 to 5, comprising culturing a recombinant host cell according to claim 8 under conditions allowing expression of the first and second human IgG CH3-containing polypeptides.

10. 1. A method for improving the stability of a heterodimeric protein comprising a first human IgG CH3-containing polypeptide comprising the amino acid mutations L351D and L368E, and a second human IgG CH3-containing polypeptide comprising the amino acid mutations T366K and L351K, wherein the method comprises introducing amino acid mutations at positions S364, K409 and / or K360 of the first human IgG CH3-containing polypeptide.

11. 1. A method for reducing the stability of a homodimeric protein comprising a first human IgG CH3-containing polypeptide comprising amino acid mutations L351D and L368E, wherein the method comprises introducing amino acid mutations at positions S364, K409 and / or K360 of the first human IgG CH3-containing polypeptide.

12. 1. A method for improving the yield of a heterodimeric protein comprising a first human IgG CH3-containing polypeptide and a second human IgG CH3-containing polypeptide, wherein said first human IgG CH3-containing polypeptide comprises amino acid mutations L351D and L368E and said second human IgG CH3-containing polypeptide comprises amino acid mutations T366K and L351K, and the method comprises introducing amino acid mutations at positions S364, K409 and / or K360 of the first human IgG CH3-containing polypeptide.

13. 1. A method for increasing the purity of a heterodimeric protein comprising a first human IgG CH3-containing polypeptide and a second human IgG CH3-containing polypeptide, wherein the first human IgG CH3-containing polypeptide comprises amino acid mutations L351D and L368E and the second human IgG CH3-containing polypeptide comprises amino acid mutations T366K and L351K, the method comprising: (a) introducing amino acid mutations at positions S364, K409 and / or K360 of the first human IgG CH3-containing polypeptide; and (b) subjecting the heterodimeric protein to ion exchange chromatography.

14. a) the amino acid at position 364 is valine, isoleucine, threonine, glutamine or leucine; and / or b) the amino acid at position 409 is isoleucine, leucine or glutamic acid; and / or c) the amino acid at position 360 is aspartic acid; 14. The method according to any one of claims 10 to 13.

15. A heterodimeric protein produced by the method of claim 9.

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