Methods and means for producing immunoglobulin-like molecules

Abstract

The present invention relates to methods and means for producing immunoglobulin-like molecules. The present invention provides means and methods for producing one or more immunoglobulin-like molecules in a single host cell. Also provided are novel CH3 mutations capable of producing target monospecific and / or bispecific immunoglobulin-like molecules.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201710637343.2 and invention name “Methods and means for producing immunoglobulin-like molecules”, which is a divisional application filed on July 28, 2017. The application to which this application is directed is a divisional application of the Chinese patent application with application number 201380032190.9 and invention name “Methods and means for producing immunoglobulin-like molecules”, which is a PCT international application PCT / NL2013 / 050293 filed on April 19, 2013 that entered the Chinese national phase. Technical Field

[0002] The present invention relates to the fields of molecular biology, medicine and biotherapeutics, and in particular to the field of therapeutic antibodies for treating various diseases. Background Art

[0003] Many currently used biotherapeutic agents are isolated recombinant human or humanized monoclonal antibodies that enhance the ability of the body's immune system to neutralize or eliminate cells and / or molecules involved in the disease process or to eliminate invading pathogens or infectious agents. Monoclonal antibodies are combined with a single specific region or epitope of an antigen, and for use in treatment, are usually selected according to desired functional properties, such as killing tumor cells, blocking receptor-ligand interactions or virus neutralization. At present, there are about 30 FDA-approved monoclonal antibodies, which are usually mass-produced, and their biophysical and biochemical properties can be analyzed in detail to ensure batch-to-batch consistency, which helps to control the qualified rate (regulatory acceptance). Despite these favorable characteristics, monoclonal antibodies also have many disadvantages, some of which are related to the complexity of their monospecific properties and diseases. Disease processes are often multifactorial in nature, and involve the numerous or synergistic effects of disease-modifying factors or the upregulation of different receptors, including the crosstalk between their signaling networks. Therefore, the blocking of a variety of different factors and pathways involved in pathology can lead to improved therapeutic effects. Due to its monospecific property, monoclonal antibodies may only interfere with a single step in a complex disease process, which often does not have optimal effects. Except for the multiple aspects that can not fully resolve the disease process, it is already clear that the single epitope of a targeted single cell protein or soluble protein or pathogen is usually not enough to effectively treat the disease, because the target epitope that is combined with it and plays the desired effect may no longer be available for a monoclonal antibody. For example, tumor cells often escape the treatment of monoclonal antibodies by lowering, mutating or shielding the target epitope present in the growth factor receptor. By activating alternative receptors and / or its ligands, tumor cells can then develop different pathways to continue to grow and transfer. Similarly, viruses and other pathogens frequently mutate and lose or shield the target epitope, thereby escaping the treatment of monoclonal antibodies. Monoclonal antibodies that bind to a single epitope do not typically recruit the full range of effector mechanisms induced by polyclonal antibodies, which include: opsonization (enhanced phagocytosis of the antigen), steric hindrance (antigen coated with the antibody is prevented from attaching to host cells or mucosal surfaces), toxin neutralization, agglutination or precipitation (aggregation and subsequent clearance caused by antibody binding to multiple soluble antigens), activation of complement, and antibody-dependent cellular cytotoxicity (antibodies enable killing of target cells by natural killer cells and neutrophils).

[0004] Polyclonal antibodies for therapeutic applications can be obtained from mixed human serum. The therapeutic polyclonal antibodies derived from this serum can be used, for example, to treat or prevent infections caused by viruses (such as rabies virus, cytomegalovirus and respiratory syncytial virus), neutralize toxins (such as tetanus toxin and botulinum toxin) or prevent Rhesus D allograft immunity. The following facts prevent the wider application of serum-derived polyclonal antibody preparations: source plasma can only be used for targets of a limited range, such as infectious diseases and toxins. In addition, in terms of both quantity and suitability, the product is highly dependent on the availability of donor blood, resulting in considerable differences between batches. In addition, screening technology cannot keep up with the evolving virus, and therefore, immunoglobulin products have the potential risk of spreading infectious diseases. Finally, the long process of blood collection, screening and immunoglobulin purification means that plasma-derived immunoglobulin production is expensive.

[0005] The mixture of monoclonal antibodies can improve the effectiveness of monoclonal antibodies, while avoiding the limitations relevant to the polyclonal antibodies derived from serum. In the art, the combination of two people or humanized monoclonal antibodies (for example, the mixture of two monoclonal antibodies for HER2 receptor, the mixture of two antibodies for EGFR receptor, two monoclonal antibodies for rabies virus) has been tested in preclinical models and clinical trials. In the art, it has been shown that the combination of two monoclonal antibodies can have an additional effect or a synergistic effect, and recruits an effector mechanism that is not related to any single antibody. For example, the mixture of two monoclonal antibodies for EGFR or HER2 shows a combination based on the following activity to kill tumor cells more effectively, and the activity includes: strengthening receptor internalization, improving the blocking of the downstream signaling pathways of receptor, and enhancing the cytotoxicity mediated by immune effectors. For the combination therapy based on 2 kinds of monoclonal antibodies, the constituent antibodies can be produced respectively and combined at the protein level. A shortcoming of this method is that the cost of developing 2 kinds of antibodies separately in clinical trials and (partially) repeating the process with a combination is huge. This causes the cost of the treatment based on antibody combination to be unacceptable. Alternatively, two recombinant cell lines producing the constituent monoclonal antibodies can be mixed in a fermenter, and the resulting antibody mixture can be purified as a single preparation (WO2004 / 061104). A disadvantage of this approach is the poor control of its composition, and therefore the poor reproducibility of the resulting recombinant polyclonal antibody preparation, especially considering that such a composition may change over time when the cells are cultured.

[0006] In the past decade, bispecific antibodies have emerged as an alternative to using a combination of two antibodies. Although, the combination of two antibodies represents a mixture of two different immunoglobulin molecules that bind to different epitopes on the same or different targets, in bispecific antibodies, this is achieved by a single immunoglobulin molecule. By binding to two epitopes on the same or different targets, bispecific antibodies can have similar effects compared to the combination of two antibodies that bind to the same epitope. In addition, in a single formulation, due to the combination of bispecific antibodies in the form of IgG with two different monovalent binding regions in a single molecule and a mixture of two IgG antibodies with two different bivalent binding molecules, different effects of these forms have also been observed. From a technical and management perspective, since manufacturing, preclinical trials and clinical trials involve a single molecule, this makes the development of a single bispecific antibody less complex. Therefore, a less complex and cost-effective drug development process while providing more effective antibody treatments promotes therapies based on single bispecific antibodies.

[0007] Bispecific antibodies based on IgG form have been produced by a variety of methods, which are composed of two heavy chains and two light chains. For example, bispecific antibodies can be produced by merging two antibody secretory cell lines to produce new cell lines or by expressing two antibodies in a single cell with recombinant DNA technology. These methods have produced a variety of antibodies, because the heavy chain from each antibody may form a monospecific dimer (also referred to as a homodimer), which comprises two identical paired heavy chains with the same specificity, and a bispecific dimer (also referred to as a heterodimer), which comprises two different paired heavy chains with different specificities. In addition, the light chain and heavy chain from each antibody can be randomly paired to form an inappropriate, non-functional combination. This problem is referred to as the mispairing of heavy chain and light chain, which can be solved by selecting an antibody with a common light chain to be expressed as bispecific. But even when using a common light chain, expressing two heavy chains and a common light chain in a single cell will also produce 3 different antibody species, i.e., 2 monospecific "parent" antibodies and the bispecific antibody, so it is necessary to purify the bispecific antibody of interest from the resulting antibody mixture. While techniques have been used to further increase the percentage of bispecific antibodies in mixtures of parent antibodies and bispecific antibodies and to reduce the percentage of heavy and light chain mispairing, there remains a need for bispecific formats that eliminate or minimize some of the disadvantages noted above.

[0008] In summary, the art provides a variety of techniques and methods for producing monoclonal antibodies, bispecific antibodies, mixtures of monoclonal antibodies, or mixtures of monospecific and bispecific antibodies that can then be used for therapeutic applications in patients. However, as discussed above, each of these existing techniques and methods has its disadvantages and limitations. Therefore, there is a need for improved and / or alternative techniques for producing biotherapeutics that target multiple disease-modifying molecules in a mixture or bispecific manner. Summary of the invention

[0009] The present invention provides methods and means for improved and / or alternative technologies for producing biotherapeutics targeting multiple disease-modifying molecules in a mixture or bispecific manner, as well as products and uses obtained from these methods and means.

[0010] Various methods are described in the art to facilitate the formation of a bispecific antibody of interest, thereby reducing the content of undesired antibodies in the resulting mixture.

[0011] For antibodies, it is well known that CH3-CH3 interactions are the main driving force for Fc dimerization (Ellerson JR et al., J. Immunol 1976 (Vol. 116) pp. 510-517; Deisenhofer J. biochemistry 1981 (Vol. 20) pp. 2361-2370). It is also well known that when two CH3 domains interact with each other, they contact at a protein-protein interface comprising "contact" residues (also referred to as contact amino acids, interface residues or interface amino acids). The contact amino acids of the first CH3 domain interact with one or more contact amino acids of the second CH3 domain. In the three-dimensional structure of an antibody, the contact amino acids are usually within 5.5 angstroms (preferably 4.5 angstroms) of each other. The interaction between the contact residues from one CH3 domain and the contact residues from a different CH3 domain may be through, 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 shown previously that approximately one-third of the contacting amino acid side chains at the CH3 domain interface of human IgG1 may be major contributors to domain folding and association. It can be further envisioned that other (neighboring) amino acid residues may influence interactions in the protein-protein interface.

[0012] Methods for interfering with antibody heavy chain dimerization have been used in the art. Specific modifications are made in the CH3 domain to favor heterodimerization over homodimerization. Examples of such modifications of the CH3-CH3 interface include the introduction of complementary protrusion mutations and cavity mutations, also known as the "knob-into-hole" method, such as described in WO1998 / 050431, Ridgeway et al. (1996) and Merchant et al. (1998).

[0013] Typically, the method includes introducing a protrusion at the interface of a first polypeptide and a corresponding cavity at the interface of a second polypeptide, such that the protrusion can be placed in the cavity, thereby promoting the formation of heteropolymers and hindering the formation of homopolymers. The "protrusion" or "knot" is constructed by replacing the small amino acid side chains at the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). A compensatory "cavity" or "hole" of the same or similar size as the protrusion is produced at the interface of the second polypeptide by replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine). The protrusions and cavities can be made by synthetic means (e.g., altering the nucleic acid encoding the polypeptide) or by peptide synthesis.

[0014] Using the knot-into-hole technique alone, the ratio of the target bispecific antibody in the mixture of two parent antibodies and the bispecific antibody is at most 87%. By introducing an additional disulfide bond between the two CH3 domains at the CH3-CH3 interface, Merchant et al. successfully increased the ratio of the bispecific antibody to 95% of the mixture. Nevertheless, in order to use such a bispecific antibody as a drug, the bispecific antibody must also be purified (separated) from the homodimer and formulated into a pharmaceutically acceptable diluent or excipient. Due to the similarity of the physicochemical properties of homodimers and heterodimers, purifying heterodimers from this mixture is a major challenge. An object of the present invention is to provide a method for producing bispecific antibodies in single cell clones and the ratio of bispecific antibodies in a mixture. Therefore, according to the present invention, the knot-into-hole technique can be used as a means (alone or together with other means) to achieve the further improved bispecific ratio in the mixture.

[0015] Another example of this transformation of the CH3-CH3 interface is provided by heterodimer Fc technology, which supports the design of bispecific and asymmetric fusion proteins by designing strand exchange engineered domain (strand-exchange engineered domain, SEED) CH3 heterodimers. These SEED CH3 heterodimers are derivatives of human IgG and IgA CH3 domains composed of alternating fragments of the CH3 sequences of human IgA and IgG, which result in the pairing of complementary human SEED CH3 heterodimers, i.e., so-called SEED-bodies (Davis JH. et al., Protein Engineering, Design & Selection 2010 (Volume 23) pp. 195-202; WO2007 / 110205).

[0016] Another approach for generating a given bispecific antibody of interest is based on the electrostatic modification of naturally charged contact residues within the CH3-CH3 interface, such as described in EP01870459 or US2010 / 0015133, WO2007 / 147901, WO2010 / 129304, Gunasekaran et al. (2010) and WO2009 / 089004. These publications describe mutations in the heavy chain CH3 domain in which naturally occurring contact residues of charged amino acids are replaced by amino acid residues of opposite charge (i.e., charge reversal strategy). This results in altered charge polarity across the Fc dimer interface, such that co-expression of electrostatically matched Fc chains supports favorable attractive interactions, thereby promoting the formation of desired Fc heterodimers, while unfavorable repulsive charge interactions inhibit the formation of undesirable Fc homodimers.

[0017] According to the description, four unique charged residues in the CH3-CH3 interface participate in the interaction of domain-domain. These are D356 / K439', E357 / K370', K392 / D399' and D399 / K409' (numbered according to Kabat (1991), wherein the residue in the first chain is separated from the residue in the second chain by " / ", and the apostrophe (') therein represents the residue numbering in the second chain). Because the CH3-CH3 interface shows secondary symmetry, each unique charge pair is represented twice in the complete IgG (that is, K439 / D356', K370 / E357', D399 / K392' and K409 / D399' charge interactions are also present in the interface). Utilizing this secondary symmetry, it has been confirmed that a single charge reversal (e.g., K409D in the first chain, or D399'K in the second chain) causes homodimers to form reductions due to the repulsion of the same charge. Combinations of different charge reversals further enhance this repulsive effect. It has been demonstrated that the expression of different CH3 domains containing different complementary charge reversals can drive heterodimerization, resulting in an increased proportion of bispecific species in the mixture.

[0018] Using the above method, bispecific antibodies with a ratio of about 76% to about 96% can be produced in a single cell. An object of the present invention is to provide a method for producing bispecific antibodies in a single cell and further improving the percentage of desired bispecific antibodies. According to the present invention, electrostatic modification technology can be used as one of the means, alone or together with other means (such as the knot-in-hole method), to achieve the further improvement of the percentage of the desired (bispecific) antibody.

[0019] In one aspect, the present invention provides a method for producing at least two different immunoglobulin-like (Ig-like) molecules from a single host cell, wherein the two immunoglobulin-like molecules each comprise two CH3 domains capable of forming an interface, the method comprising providing in the cell:

[0020] a) a first nucleic acid molecule encoding a first CH3 domain-comprising polypeptide chain,

[0021] b) a second nucleic acid molecule encoding a second CH3 domain-containing polypeptide chain,

[0022] c) a third nucleic acid molecule encoding a third CH3 domain-containing polypeptide chain, and

[0023] d) a fourth nucleic acid molecule encoding a fourth CH3 domain-containing polypeptide chain,

[0024] wherein at least two of the nucleic acid molecules have means for preferentially pairing the first CH3 domain-containing polypeptide chain with the second CH3 domain-containing polypeptide and the third CH3 domain-containing polypeptide with the fourth CH3 domain-containing polypeptide, the method further comprising the steps of culturing the host cell and allowing the at least four nucleic acid molecules to be expressed and harvesting the at least two different immunoglobulin-like molecules from the culture.

[0025] It is often desirable to generate more than one (bispecific) antibody in order, for example, to more effectively interfere with multiple biological pathways involved in a disease process or to interfere with the entry, replication and / or spread of a pathogen.

[0026] The mixture of more than a bispecific antibody is also particularly useful for the treatment of certain diseases.For example, in the treatment process with antibody or small molecule drugs, tumor cells use many different strategies to develop resistance.Resistance may relate to multiple cell surface receptors and soluble molecules, and it is considered to be conducive to the development of a variety of such disease-related molecules and escape from the antibody-based cancer therapy of related molecules.In the case of comprising more than 2 such disease-related target molecules or epitopes and escape from related target molecules or epitopes, the mixture of bispecific antibodies provides an innovative and attractive treatment form.Preferably, the mixture of this bispecific antibody produced by a single cell is conducive to the drug development process, that is, complexity is lower from a management perspective, cost-effective and feasible from the perspective of drug manufacturing and clinical development.In the method based on a single cell, it is desirable to use a method that can be controlled and effectively produce bispecific antibodies, thereby reducing or even completely eliminating the demand for separating the mixture of desired bispecific IgG molecules from the IgG molecule of undesirable monospecificity. In the prior art, mixtures of monospecific antibodies and bispecific antibodies have been produced by single cells (WO2004 / 009618), but these mixtures represent complex mixtures of a variety of different bispecific and monospecific antibody species. Another object of the present invention is to provide means and methods for producing a defined mixture of bispecific antibodies in a single cell. Preferably, the method provided is such that the mixture of (bispecific) antibodies accounts for at least 95%, at least 97% or even more than 99% of dimeric IgG molecules, regardless of the amount of monomer by-products, see herein below. Typically, in cells producing multiple complete IgG molecules, there may be half molecules (monomer by-products) that can be simply removed by size exclusion chromatography as known in the art.

[0027] In one embodiment, the present invention provides a method for producing a limited mixture of at least two different immunoglobulin-like molecules in a single cell instead of a single purpose (bispecific) antibody, wherein the formation of other undesirable dimeric antibody species is reduced or even absent. The resulting mixture is well defined, and its composition is controlled by the design of the CH3 domain mutant. In addition, the expression level and / or the regulation of different transfection ratios for expression affect the composition of the mixture. In the method according to the present invention, the first nucleic acid molecule encodes a CH3 domain that is preferentially paired with a CH3 domain encoded by a second nucleic acid molecule, and the third nucleic acid molecule encodes a CH3 domain that is preferentially paired with a CH3 domain encoded by a fourth nucleic acid molecule. The present invention also provides a mixture of at least two different immunoglobulin-like molecules that can be obtained by the method of the present invention.

[0028] As used herein, the term "preferential pairing of the first CH3 domain-containing polypeptide and the second CH3 domain-containing polypeptide" means that substantially all resulting dimers comprising the first CH3 domain-containing polypeptide and / or the second CH3 domain-containing polypeptide are dimers consisting of one first CH3 domain-containing polypeptide paired with one second CH3 domain-containing polypeptide. Similarly, the term "preferential pairing of the third CH3 domain-containing polypeptide and the fourth CH3 domain-containing polypeptide" means that substantially all resulting dimers comprising the third CH3 domain-containing polypeptide and / or the fourth CH3 domain-containing polypeptide are dimers consisting of one third CH3 domain-containing polypeptide paired with one fourth CH3 domain-containing polypeptide. As a result, when nucleic acid molecules encoding four different (A, B, C, D) CH3 domain-containing polypeptides are introduced into a single cell, a mixture of mainly two specific immunoglobulin-like molecules is produced, rather than a mixture of 10 different immunoglobulin-like dimers (AA, AB, AC, AD, BB, BC, BD, CC, CD, and DD).

[0029] As explained in more detail below, in a preferred embodiment, the first CH3 domain-containing polypeptide chain comprises the amino acid substitution T366K, and the second CH3 domain-containing polypeptide chain comprises the amino acid substitution L351D. These amino acid changes are preferred means for preferentially pairing the first CH3 domain-containing polypeptide chain with the second CH3 domain-containing polypeptide chain. The first CH3 domain-containing polypeptide chain preferably further comprises the amino acid substitution L351K. Furthermore, the second CH3 domain-containing polypeptide chain preferably further comprises an amino acid substitution selected from the group consisting of Y349E, Y349D and L368E, most preferably L368E. In another preferred embodiment, the third CH3 domain-containing polypeptide chain comprises the amino acid substitutions E356K and D399K, and the fourth CH3 domain-containing polypeptide chain comprises the amino acid substitutions K392D and K409D.

[0030] In the method according to the present invention, each polypeptide chain containing a CH3 domain preferably also includes a variable region that recognizes a target epitope. The variable region as a part of a polypeptide chain containing a CH3 domain preferably has a common light chain. In this case, only the VH of the variable region is different, while the VL in all variable regions is substantially the same. Therefore, in a preferred aspect, a method according to the present invention is provided, which also includes providing a nucleic acid molecule encoding a common light chain to the host cell. In a particularly preferred embodiment, the four variable regions of the four polypeptide chains containing CH3 domains each recognize a different target epitope. For example, if the heavy chain encoded by the first nucleic acid molecule also includes a variable domain specific for antigen A, the heavy chain encoded by the second nucleic acid molecule also includes a variable domain specific for antigen B, the heavy chain encoded by the third nucleic acid molecule also includes a variable domain specific for antigen C, and the heavy chain encoded by the fourth nucleic acid molecule also includes a variable domain specific for antigen D, then a mixture of bispecific immunoglobulin-like molecules containing AB specificity and bispecific immunoglobulin-like molecules containing CD specificity will be produced. Due to the means for preferential pairing of the first CH3 domain-containing polypeptide with the second CH3 domain-containing polypeptide and the third CH3 domain-containing polypeptide with the fourth CH3 domain-containing polypeptide, the formation of monospecific antibodies (with AA, BB, CC or DD specificity) or bispecific antibodies with AC, AD, BC or BD specificity is reduced or even absent. Of course, additional nucleic acid molecules, such as nucleic acid molecules encoding the fifth CH3 domain-containing polypeptide and the sixth CH3 domain-containing polypeptide, can be used to generate a defined mixture comprising more than two different immunoglobulin-like molecules.

[0031] It is noteworthy that the ratio of the nucleic acids used in the method according to the present invention is not necessarily 1: 1: 1: 1, and the ratio of the immunoglobulin-like molecules expressed is not necessarily 1: 1. Antibody mixtures with optimal ratios can be produced using means known in the art. For example, by using different genetic elements (e.g., promoters, enhancers, and repressors) or by controlling the copy number of the genomic integration site of the DNA construct encoding the antibody, the expression level of the nucleic acid molecules can be adjusted and the ratio of the immunoglobulin-like molecules produced can be adjusted accordingly.

[0032] The means for preferential pairing may preferably include modified complementary knot-into-hole mutations, disulfide bonds, charge mutations (including charge reversal mutations), or combinations thereof. It will be appreciated by those skilled in the art that the means for preferential pairing may be selected among certain types of mutations, i.e., all at least 4 nucleic acid molecules encoding polypeptide chains containing CH3 domains may all contain, for example, charge mutations as means for preferential pairing. Additionally, in some cases, unmodified wild-type CH3 may also be used to preferentially pair two wild-type polypeptide chains containing CH3 domains. In a particularly preferred embodiment, the means for preferential pairing include at least one CH3 mutation selected from Table B, as explained in other parts of this application. Therefore, a preferred embodiment provides a method according to the present invention, wherein all four nucleic acid molecules have means for preferentially pairing the first CH3 domain-containing polypeptide with the second CH3 domain-containing polypeptide and the third CH3 domain-containing polypeptide with the fourth CH3 domain-containing polypeptide, wherein the means for preferentially pairing the first CH3 domain-containing polypeptide with the second CH3 domain-containing polypeptide are different from those for preferentially pairing the third CH3 domain-containing polypeptide with the fourth CH3 domain-containing polypeptide.

[0033] One aspect of the present invention provides a method according to the present invention, wherein the means for preferentially pairing the first CH3 domain-containing polypeptide with the second CH3 domain-containing polypeptide is different from the means for preferentially pairing the third CH3 domain-containing polypeptide with the fourth CH3 domain-containing polypeptide. "Different from" means that the means for preferentially pairing the first CH3 domain-containing polypeptide with the second CH3 domain-containing polypeptide is designed so as to favor preferential pairing of the first chain with the second chain. The design is such that substantially no interaction occurs between the first CH3 domain-containing polypeptide chain and the third and / or fourth CH3 domain-containing polypeptide chains. In other words, dimerization between the first CH3 domain-containing polypeptide and the third polypeptide or the fourth polypeptide is reduced to substantially zero, etc. The third CH3 domain-containing polypeptide and the fourth CH3 domain-containing polypeptide may be wild-type or may comprise means for preferential pairing, wherein the means are different from the means for preferential pairing the first CH3 domain with the second CH3 domain. Current research focuses on the use of, for example, the knob-into-hole technique or mutating (reversing) the charged contact amino acids present in the CH3 domain to produce a single bispecific antibody. However, prior to the present invention, it has not been possible to produce a defined mixture of at least two (bispecific) immunoglobulin-like molecules without significant co-production of other dimer byproducts.

[0034] The present invention provides methods for efficiently and controllably producing mixtures of well-defined immunoglobulin-like molecules with a high ratio of bispecifics in the mixture. In systems where two bispecifics are required, even ratios of (two) bispecifics of at least 95%, at least 97% or more are obtained. This means that only at most 5%, at most 3% or less monospecific bivalent byproducts are obtained. It is worth noting that the amount of monomer byproducts (i.e. half molecules) is not so important, because these half molecules can be easily separated from dimers by virtue of their size difference.

[0035] In another preferred embodiment, the variable regions of the first and second CH3 domain-containing polypeptide chains recognize different target epitopes, while the variable regions of the third and fourth CH3 domain-containing polypeptide chains recognize the same target epitope. This will result in the production of primarily one bispecific immunoglobulin-like molecule and one monospecific immunoglobulin-like molecule. For example, if the variable regions of the first and second CH3 domain-containing polypeptide chains recognize different target epitopes, and if the variable regions of the third and fourth CH3 domain-containing polypeptide chains both recognize the same target epitope that is different from the target epitope recognized by the first and second CH3 domains, a mixture of immunoglobulin-like molecules with AB or CC specificity will be formed. Therefore, a method according to the present invention is also provided, wherein the target epitope recognized by the variable regions of the third and fourth CH3 domain-containing polypeptide chains is the same, but different from the target epitope recognized by the variable regions of the first or second CH3 domain-containing polypeptide chains.

[0036] Alternatively, when the variable regions of the first and second CH3 domain-containing polypeptide chains recognize different target epitopes, and when the variable regions of the third and fourth CH3 domain-containing polypeptide chains both recognize the same epitope as the first or second CH3 domain-containing polypeptide chain, a mixture of immunoglobulin-like molecules with AB and AA, or AB and BB specificities will be formed. Thus, a method according to the invention is provided, wherein the target epitope recognized by the variable regions of the third and fourth CH3 domain-containing polypeptide chains is the same as the target epitope recognized by the variable regions of the first or second CH3 domain-containing polypeptide chain. Another object of the present invention is to provide a means and method for producing a defined mixture of bispecific antibodies and monospecific antibodies in a single cell culture. A non-limiting example of such a well-defined mixture is a mixture of a bispecific antibody with AB specificity and a monospecific antibody with AA specificity. Another example is a mixture of a bispecific antibody with AB specificity and a monospecific antibody with BB specificity. Another example is a mixture of a bispecific antibody with AB specificity and a monospecific antibody with CC specificity. Additionally, preferred means and methods are provided which produce a mixture in which the antibody of interest is at least 90%, more preferably at least 95% and most preferably at least 97% or even more than 99% of the desired antibody.

[0037] In another embodiment, a method according to the invention is provided, wherein the variable regions of the first and second CH3 domain-containing polypeptide chains recognize the same target epitope, and the variable regions of the third and fourth CH3 domain-containing polypeptide chains recognize a second target epitope, which is different from the target epitope recognized by the first and second variable regions. This will result in the production of monospecific immunoglobulin-like molecules with AA specificity or BB specificity. The formation of bispecific immunoglobulin-like molecules is reduced or even avoided. In multiple embodiments, it is preferred to produce a mixture of monospecific antibodies in a single cell rather than a mixture of bispecific antibodies. For example, when two identical target molecules need to be cross-linked, or when two targets are too far apart to be bound by a single bispecific antibody. It may also be advantageous to produce a mixture of monospecific antibodies in a single cell because the mixture can be regarded as a single therapeutic product. In the art, the therapeutic effects and safety of a variety of monospecific antibodies have been demonstrated and market authorization has been obtained. Producing a mixture of monospecific antibodies in a single cell will therefore be conducive to testing the effects and safety of several such mixtures, and will reduce the effort and cost of registration approval and manufacturing. However, there is currently no method available for producing a specific mixture of monospecific antibodies in a single cell and wherein the formation of bispecific side products is reduced to less than 5%. Another object of the present invention is to provide means and methods for producing such a well-defined homodimeric antibody mixture in a single cell and wherein the formation of bispecific antibodies is reduced to less than 5%.

[0038] Thus, a method according to the invention is suitable for producing any desired mixture of bispecific and / or monospecific immunoglobulin-like molecules. In addition, additional nucleic acid molecules, such as nucleic acid molecules encoding the fifth and sixth (and seventh and eighth, etc.) CH3 domain-containing polypeptides, can be used to produce defined mixtures containing more than two different immunoglobulin-like molecules.

[0039] Preferably, in a method according to the invention, at least two CH3 domains are used, which comprise at least one combination of mutations provided by the invention. By means of these mutations, new specific interactions are formed between the two CH3 domains. These mutations according to the invention will be discussed in more detail below.

[0040] The term "immunoglobulin-like molecule" as used herein refers to a protein molecule having at least one immunoglobulin (Ig) domain. The immunoglobulin-like molecule comprises a sequence having the function of at least an immunoglobulin CH3 domain, preferably a sequence comprising the CH3 domain of IgG1. The protein molecule having at least one CH3 domain can be further equipped with a specific binding portion. Therefore, the CH3 domain of the present invention comprising a means for preferential pairing can be used to preferentially pair two protein molecules containing CH3 domains to design the desired heterodimeric binding molecules or mixtures of binding molecules. The portion that binds to the protein molecule containing the CH3 domain can be designed to be any binding agent, including but not limited to single-chain Fvs, single chains or tandem diabodies. VHH, Fab, ankyrin repeat protein or DART, TCR-like antibodies, MicroProteins or In a preferred embodiment, the binding moiety is an antibody variable region (i.e., a VH / VL combination). The variable regions that are part of the polypeptide chain containing the CH3 domain preferably have a common light chain. In this case, only the VH of the variable region is different, while the VL of all variable regions are essentially the same.

[0041] Alternatively or additionally, other molecules may be engineered into the CH3 domains of the invention, including cytokines, hormones, soluble ligands, receptors and / or peptides.

[0042] In a more preferred embodiment, the immunoglobulin-like molecule comprises a full-length Fc skeleton. In a most preferred embodiment, the immunoglobulin-like molecule is an antibody. The variable regions of these antibodies preferably have a common light chain, but their VH regions may be different. The term "antibody" as used herein refers to a protein molecule belonging to the immunoglobulin protein class, which contains one or more domains that bind to an epitope on an antigen, wherein such domains are from the variable region of an antibody or have sequence homology with the variable region of an antibody. Antibodies are known in the art and include a variety of isotypes, such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE and IgM. The antibody according to the present invention can be any of these isotypes, or a functional derivative thereof and / or a fragment thereof. In a preferred embodiment, the immunoglobulin-like molecule produced is an antibody of the IgG isotype, because IgG antibodies, for example, have a longer half-life than antibodies of other isotypes.

[0043] The antibodies produced using the methods according to the invention can have sequences of any origin, including mouse and human sequences. Antibodies can be composed of sequences from only one source, such as fully human antibodies, or they can have sequences from more than one source, such as producing chimeric antibodies or humanized antibodies. Antibodies for therapeutic use are preferably natural antibodies of the subject to be treated as much as possible (e.g., human antibodies for human subjects). The binding of antibodies can be expressed in terms of specificity and affinity. Specificity determines which antigen or epitope thereof binds to the binding domain. Affinity is a measure of the strength of binding to a specific antigen or epitope. Specific binding is defined as having at least 1×10 -5 M, more preferably 1×10 -7 M, more preferably higher than 1×10 -g The affinity of M (K D ) binding. Typically, monoclonal antibodies for therapeutic applications have a binding affinity of up to 1×10 -10 M or even higher affinity. The term "antigen" as used herein refers to a substance or molecule that triggers the production of antibodies by the immune system when it is introduced into the body. Antigens, etc. can be derived from pathogenic organisms, tumor cells or other abnormal cells, can be derived from haptens, or even its own structure. At the molecular level, the characteristic of an antigen is that it can bind to the antigen binding site of an antibody. A mixture of antigens can also be regarded as an "antigen", that is, it should be understood by those skilled in the art that sometimes the lysate of tumor cells or viral particles can be expressed as "antigens", but there are many antigenic determinants in such tumor cell lysates or viral particle preparations. Antigens contain at least one, but often more epitopes. The term "epitope" as used herein refers to an antigenic portion that is recognized by the immune system, particularly antibodies, B cells, or T cells. Although epitopes are generally considered to be derived from non-self proteins, recognizable sequences derived from hosts can also be considered to be classified as epitopes.

[0044] The term "CH3 domain" is well known in the art. The structure of IgG has four chains, 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 chain are referred to as the Fc (fragment crystallizable) portion, the Fc fragment, the Fc backbone, or simply Fc. The IgG molecule is a heterotetramer with two heavy chains and two light chains, wherein the heavy chains are held together by disulfide bonds (-SS-) at the hinge region. The heavy chains dimerize by interactions at the CH3-CH3 domain interface and by interactions at the hinge region. The number of hinge disulfide bonds varies among immunoglobulin subclasses (Papadea and Check 1989). The Fc fragment of an immunoglobulin molecule is a dimer of the two C-terminal constant regions of the heavy chain, i.e., the CH2 and CH3 domains. Its physiological function is to interact with the complement system and with specific receptors on the surface of various cells, etc. It is known that the interaction between the CH3 domains of two separate heavy chains plays an important role in driving heavy chain dimerization. Therefore, the CH3 domain guides the association of antibody heavy chains, and it is known that the interface between the CH3 domains contains more than 20 contact residues from each chain, which play a role in CH3-CH3 interactions (Deisenhofer J., Biochemistry 1981 (Vol. 20) pp. 2361-2370; Miller S., J. Mol. Biol. 1990 (Vol. 216) pp. 965-973; Padlan, Advances in Protein Chemistry 1996 (Vol. 49) pp. 57-133). The CH3 variants of the present invention can therefore be used to associate with other antibody domains to produce bispecific or monospecific full-length antibodies. The specificity of the antibody defined by the VH / VL combination generally does not affect the dimerization behavior of the heavy chain driven by the CH3 domain.

[0045] As used herein, the terms "contact residue", "contact amino acid", "interface residue" and "interface amino acid" generally refer to any amino acid residue present in a CH3 domain that can participate in interdomain contacts, as can be calculated by techniques known in the art, including calculation of the solvent accessible surface area (ASA) of the CH3 domain residues in the presence and absence of a second chain (Lee and Richards J. Mol. Biol. 1971 (55) 379), wherein the difference in ASA between these two calculations is shown Residues at positions 347, 349, 350, 351, 352, 353, 354, 355, 356, 357, 360, 364, 366, 368, 370, 390, 392, 394, 395, 397, 399, 400, 405, 407, 409, 439 are identified as contact residues according to the EU numbering system (Table A).

[0046] Table A: List of CH3 domain interface residues

[0047] Interface residues in chain A Contact residues in chain B Q347 K360 Y349 S354, D356, E357, K360 T350 S354, R355 L351 L351, P352, P353, S354, T366 S354 Y349, T350, L351 R355 T350 D356 Y349, K439 E357 Y349, K370 K360 Q347, Y349 S364 L368, K370 T366 L351, Y407 L368 S364, K409 K370 E357, S364 N390 S400 K392 L398, D399.S400, F405 T394 T394, V397, F405, Y407 P395 V397 V397 T394, P395 D399 K392, K409 S400 N390, K392 F405 K392, T394, K409 Y407 T366, T394, Y407, K409 K409 L368, D399, F405, Y407 K439 D356

[0048] The contact residues in the CH3-CH3 interface can be charged amino acids, or neutral amino acid residues. The term "charged amino acid residue" or "charged residue" as used herein refers to an amino acid residue with a charged side chain. These can be positively charged side chains, such as those present in arginine (Arg, R), histidine (His, H) and lysine (Lys, K), or can be negatively charged side chains, such as those present 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 carry 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).

[0049] As used herein, the term "CH3-CH3 domain interface" or "CH3 interface", "CH3-CH3 pairing", "domain interface" or simply "interface" refers to the association between two CH3 domains of isolated CH3 domain-containing polypeptides, which is the result of the interaction of amino acid residues, i.e., at least one interaction between an amino acid of a first CH3 domain and an amino acid of a second CH3 domain. Such interactions are, for example, through van der Waals forces, hydrogen bonds, water-mediated hydrogen bonds, salt bridges or other electrostatic forces, attractive interactions between aromatic side chains, the formation of disulfide bonds, or other forces known to those skilled in the art.

[0050] As used herein, the means for preferentially pairing the first CH3 domain-containing polypeptide with the second CH3 domain-containing polypeptide and the third CH3 domain-containing polypeptide with the fourth CH3 domain-containing polypeptide may be any means known in the art. In one embodiment, at least one nucleic acid molecule encodes a CH3 domain containing a large amino acid residue (i.e., a "knot" or "protrusion") (e.g., R, F, Y, W, I or L) at the contact residue position, and at least one additional nucleic acid molecule encodes a CH3 domain containing a small amino acid residue (i.e., a "hole" or "cavity") (e.g., G, A, S, T or V) at the complementary contact residue position. Due to the spatial conformation of the contacting amino acids, the resulting CH3 domains will preferentially pair with each other. The knot-into-hole technique has been described in detail herein before. In another embodiment of the invention, at least one nucleic acid molecule encodes a CH3 domain comprising an amino acid now carrying an opposite charge to wild type at a naturally charged contact residue position (i.e., a naturally occurring K, H, R, D, or E), and at least one additional nucleic acid molecule encodes a CH3 domain comprising an amino acid now carrying an opposite charge to wild type at a naturally charged complementary contact residue. Due to the opposite charge of the contact amino acids, the resulting engineered CH3 domains will preferentially pair with each other, while pairing of identical CH3 domains will be reduced due to electrostatic repulsion. In one embodiment, CH3 mutations as described in EP01870459, WO2009 / 089004, Gunasekaran et al. (2010) are used. In one embodiment, the means for preferentially pairing the first CH3 domain-containing polypeptide with the second CH3 domain-containing polypeptide are "knot" and "hole" amino acid residues, while the means for preferentially pairing the third CH3 domain-containing polypeptide with the fourth CH3 domain-containing polypeptide are charge engineered amino acids. Preferably, both of said means for preferentially pairing said first CH3 domain-containing polypeptide with said second CH3 domain-containing polypeptide and preferentially pairing said third CH3 domain-containing polypeptide with said fourth CH3 domain-containing polypeptide are charge engineered amino acids. In one embodiment, different amino acid residues are engineered for preferentially pairing said first CH3 domain-containing polypeptide with said second CH3 domain-containing polypeptide than the amino acid residues engineered for preferentially pairing said third CH3 domain-containing polypeptide with said fourth CH3 domain-containing polypeptide. In a particularly preferred embodiment, at least the first nucleic acid molecule and the second nucleic acid molecule encode a CH3 domain having a novel mutation as provided by the present invention. As described in more detail below, the present invention provides novel mutations of CH3 that are capable of producing certain bispecific immunoglobulin-like molecules of interest without significant amounts of undesirable (dimer) byproducts.The present invention also provides novel CH3 mutations that are capable of producing certain desired monospecific immunoglobulin-like molecules without significant amounts of undesirable (dimer) byproducts. Therefore, it is preferred to use at least one of these CH3 mutations according to the present invention.

[0051] The terms "polypeptide", "polypeptide molecule" or "polypeptide chain" as used herein refer to a chain of amino acids covalently linked together by peptide bonds. Proteins are usually composed of one or more polypeptide molecules. Each polypeptide has a free amino group at one end, which is called the amino terminus or N-terminus. The other end with its free carboxyl group is called the carboxyl terminus or C-terminus. The polypeptide according to the present invention may have undergone post-translational modification processes and may, for example, be glycosylated. The polypeptide chain containing the CH3 domain of the present invention thus refers to a polypeptide chain that at least encompasses the Ig CH3 domain, and which may have undergone post-translational modification processes.

[0052] The term "nucleic acid molecule" as used herein is defined as a molecule comprising a nucleotide chain, more preferably a DNA and / or RNA chain. In one embodiment, double-stranded RNA is used. In other embodiments, the nucleic acid molecules of the present invention comprise other types of nucleic acid structures, for example, DNA / RNA helices, peptide nucleic acids (PNA), locked nucleic acids (LNA) and / or ribozymes. Therefore, the term "nucleic acid molecule" also encompasses chains comprising non-natural nucleotides, modified nucleotides and / or non-nucleotide building blocks that exhibit the same functions as natural nucleotides.

[0053] The present invention also provides a method for preparing a host cell for producing at least two different immunoglobulin-like molecules, the method comprising the steps of: introducing into the host cell nucleic acid sequences encoding at least first, second, third and fourth CH3 domain-containing polypeptide chains, wherein at least two of the nucleic acid sequences have means for preferentially pairing the first CH3 domain-containing polypeptide with the second CH3 domain-containing polypeptide and the third CH3 domain-containing polypeptide with the fourth CH3 domain-containing polypeptide, wherein the nucleic acid sequences are introduced sequentially or simultaneously.

[0054] Another aspect of the present invention is to provide a method for preparing a heterodimeric immunoglobulin-like molecule, the method comprising the steps of: introducing into the host cell a nucleic acid sequence encoding at least a first and a second CH3 domain-containing polypeptide chain, wherein the first CH3 domain-containing polypeptide chain comprises at least one neutral amino acid residue replaced by a positively charged amino acid residue, and wherein the second CH3 domain-containing polypeptide chain comprises at least one neutral amino acid residue replaced by a negatively charged amino acid residue, wherein the nucleic acid sequences are introduced sequentially or simultaneously. The method for preparing the host cell preferably also comprises the step of introducing into the host cell a nucleic acid sequence encoding a common light chain.

[0055] The present invention also provides a recombinant host cell comprising nucleic acid sequences encoding at least a first, second, third and fourth CH3 domain-containing polypeptide chain, wherein at least two of the nucleic acid molecules have means for preferentially pairing the first CH3 domain-containing polypeptide with the second CH3 domain-containing polypeptide and preferentially pairing the third CH3 domain-containing polypeptide with the fourth CH3 domain-containing polypeptide. The present invention also provides a recombinant host cell comprising nucleic acid sequences encoding at least a first and a second CH3 domain-containing polypeptide chain, wherein the first CH3 domain-containing polypeptide chain comprises a replacement of at least one neutral amino acid residue with a positively charged amino acid residue, and wherein the second CH3 domain-containing polypeptide chain comprises a replacement of at least one neutral amino acid residue with a negatively charged amino acid residue.

[0056] The recombinant host cell according to the present invention preferably further comprises a nucleic acid sequence encoding a common light chain.

[0057] According to the present invention, the "host cell" can be any host cell capable of expressing a recombinant DNA molecule, including: bacteria, such as Escherichia (such as Escherichia coli), Enterobacter, Salmonalla, Bacillus, Pseudomonas, Streptomyces; yeast, such as Saccharomyces cerevisiae, Kluyveromyces lactis, Pichia pastoris, Candida or Yarrowia; filamentous fungi (such as Neurospora, Aspergillus oryzae, Aspergillus nidulans and Aspergillus niger); insect cells, such as Spodoptera frugiperda, Spodoptera spp. frugiperda) SF-9 or SF-21 cells; and preferred mammalian cells, such as Chinese hamster ovary (CHO) cells, BHK cells, mouse cells including SP2 / 0 cells and NS-0 myeloma cells; primate cells, such as COS and Vero cells, MDCK cells, BRL 3A cells, hybridomas, tumor cells, immortalized primary cells; human cells, such as W138, HeDG2, HeLa, HEK293, HT1080 or embryonic retinal cells such as PER.C6, etc. Typically, the expression system selected will include a mammalian cell expression vector and a host so that the antibody can be appropriately glycosylated. Human cell lines (preferably PER.C6) can be advantageously used to obtain antibodies with a complete human glycosylation pattern. Conditions for cell growth or propagation (see, e.g., Tissue Culture, Academic Press, Kruse and Paterson, eds. (1973)) and the conditions used to express the recombinant product may vary somewhat, and the process is usually optimized to increase the ratio of the product and / or the growth of the cells relative to each other according to methods generally known to those skilled in the art. In general, the principles, protocols and practical techniques for maximizing the productivity of mammalian cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach (M. Butler ed., IRL Press, 1991). Expression of antibodies in recombinant host cells has been widely described in the art (see, e.g., EP0120694, EP0314161, EP0481790, EP0523949; U.S. Pat. No. 4,816,567, WO00 / 63403).The nucleic acid molecules encoding the light and heavy chains may be present as extrachromosomal copies and / or stably integrated into the chromosome of the host cell, the latter being preferred.

[0058] Another aspect of the present invention provides a culture of a recombinant host cell according to the present invention, or a culture of a recombinant host cell obtainable or obtained by a method according to the present invention, which produces at least two different immunoglobulin-like molecules or produces heterodimeric immunoglobulin-like molecules.

[0059] In order to obtain expression of the nucleic acid sequence encoding the polypeptide containing the CH3 domain, it is well known to those skilled in the art that a sequence capable of driving such expression can be functionally linked to the nucleic acid sequence encoding the polypeptide containing the CH3 domain. Functional linkage is intended to describe: linking the nucleic acid sequence encoding the polypeptide containing the CH3 domain or its precursor to a sequence capable of driving expression, so that these sequences can drive the expression of the polypeptide containing the CH3 domain or its precursor. Useful expression vectors are available in the art, such as Invitrogen's pcDNA vector series. In which the sequence encoding the target polypeptide is correctly inserted relative to the sequence controlling the transcription and translation of the encoded polypeptide, the resulting expression cassette can be used to produce the target polypeptide, which is called expression. The sequences driving expression can include promoters, enhancers, etc., and combinations thereof. These should be able to function in the host cell to drive the expression of the nucleic acid sequence functionally linked to them. Promoters can be constitutive or regulated, and can be obtained from a variety of sources, including viruses, prokaryotic or eukaryotic sources, or artificially designed. The expression of the target nucleic acid can come from a natural promoter or its derivatives, or from a completely heterologous promoter. Some well-known and very commonly used promoters for expression in eukaryotic cells include promoters from viruses (such as adenoviruses) such as E1A promoters, promoters from cytomegalovirus (CMV) such as CMV immediate early (IE) promoters, promoters from Simian Virus 40 (SV40), and the like. Suitable promoters may also be from eukaryotic cells, such as metallothionein (MT) promoters, elongation factor 1α (EF-1α) promoters, actin promoters, immunoglobulin promoters, heat shock promoters, and the like. Any promoter or enhancer / promoter capable of driving expression of a sequence of interest in a host cell is suitable for use in the present invention. In one embodiment, the sequence capable of driving expression comprises a region from a CMV promoter, preferably a region from nucleotides -735 to +95 of a CMV immediate early gene enhancer / promoter. Those skilled in the art will recognize that the expression sequences used in the present invention may be appropriately combined with elements capable of stabilizing or enhancing expression (e.g., insulators, matrix attachment regions, STAR elements (WO03 / 004704), and the like). This may enhance stability and / or expression levels.

[0060] The production of proteins in recombinant host cells has been widely 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. The cells are cultured so that they can metabolize and / or grow and / or divide and / or produce the desired recombinant protein. This can be accomplished by methods known to those skilled in the art, including but not limited to providing nutrients to the cells. The method includes growth attached to a surface, growth in suspension, or a combination thereof. Multiple culture conditions can be optimized by methods known in the art to optimize protein yield. Cultivation can be carried out in, for example, a culture dish, a roller bottle, or a bioreactor, using batch, fed-batch, continuous system, hollow fiber, etc. In order to achieve large-scale (continuous) production of recombinant proteins by cell culture, it is preferred in the art to enable the cells to grow in suspension, and preferably to enable the cells to be cultured in the absence of serum from an animal or human source or serum components from an animal or human source. The absence of additional animal or human proteins from the culture medium allows for easier purification and increased safety, while the system is also reliable as the synthetic medium provides the best reproducibility.

[0061] Immunoglobulin-like molecules are expressed in host cells and harvested from cells or preferably from cell culture medium by methods commonly known to those skilled in the art. After harvesting, these immunoglobulin-like molecules can be purified using methods known in the art. Such methods may include precipitation, centrifugation, filtration, size exclusion chromatography, affinity chromatography, cation and / or anion exchange chromatography, hydrophobic interaction chromatography, etc. For mixtures comprising antibodies of IgG molecules, protein A or protein G, affinity chromatography (see, for example, U.S. Patents 4,801,687 and 5,151,504) may be suitably used.

[0062] The immunoglobulin-like molecules and / or mixtures thereof produced according to the method of the present invention preferably have a common light chain. Therefore, a method according to the present invention is also provided, which also includes providing a nucleic acid molecule encoding a common light chain to the host cell. This is a light chain that can be paired with at least two different heavy chains to form a functional antigen-binding domain. The functional antigen-binding domain can specifically bind to an antigen. Preferably, a common light chain that can be paired with all heavy chains produced according to the method of the present invention to form a functional antigen-binding domain is used, so that the mispairing of unmatched heavy chains and light chains is avoided. In one aspect, only a common light chain with a same amino acid sequence is used. Alternatively, those skilled in the art will recognize that "common" also refers to a functionally equivalent light chain with different amino acid sequences. There are many variants of the light chain, in which there are mutations (deletions, substitutions, additions) that do not substantially affect the formation of the functional binding region. Such variants are therefore also able to bind to different heavy chains and form functional antigen-binding domains. Therefore, the term "common light chain" used herein refers to a light chain that may be identical or has some amino acid sequence differences but the resulting antibody retains a light chain of binding specificity after pairing with the heavy chain. For example, such as by introducing and testing conservative amino acid changes and / or amino acid changes in regions that do not or only partially help binding specificity when paired with a heavy chain, it is possible to prepare or find light chains that are not identical but still functionally equivalent. The combination of certain common light chains and such functionally equivalent variants is encompassed in the term "common light chain". Reference may be made to the detailed description of the usage of common light chains in WO2004 / 009618. Preferably, the common light chain used in the present invention is a germline sample light chain, more preferably a germline light chain, preferably a rearranged germline human κ light chain, most preferably a rearranged germline human κ light chain IgVκ1-39 / Jκ or IGVκ3-20 / Jκ.

[0063] Alternatively, the skilled person may choose to employ means for forced pairing of heavy and light chains as an alternative to using a common light chain and avoiding mispairing of unmatched heavy and light chains, such means being described in, for example, WO2009 / 080251, WO2009 / 080252 and / or WO2009 / 080253.

[0064] The present invention provides new modified CH3 domains, as well as new combinations of CH3 mutations. Prior to the present invention, the charged contact amino acids of the CH3 domain known to be involved in CH3-CH3 pairing were replaced by amino acids of opposite charge (charge reversal), thereby affecting CH3-CH3 pairing. The mutation according to the present invention is a creative alternative to this method, because now the uncharged or neutral CH3 amino acids in the wild-type CH3 are replaced by charged residues. In this embodiment, the present invention does not exchange the charged contact amino acids by amino acids of opposite charge, but replaces the uncharged CH3 amino acids with charged CH3 amino acids. The method of the present invention not only provides a method for effectively manipulating the dimerization of the CH3 domain, but also has the advantage of generating at least one additional charge-charge interaction at the CH3 interface. In view of this additional charge-charge interaction in addition to the existing charge pairs at the CH3-CH3 interface, the dimer according to the present invention is generally more stable than the wild-type dimer (the wild-type dimer is defined as a bispecific IgG (AB) without CH3 modification compared to its parent homodimer (AA or BB)). In addition, it has been surprising that the proportion of one or more target immunoglobulin-like molecules in the mixture can be further increased. As mentioned above, known methods in the art for preferentially producing bispecific antibodies generally include the production of some undesirable dimerization byproducts. For example, the proportion of target bispecific antibodies using the knot-into-hole technology is at most 87%, while the electrostatic transformation method in which the charged contacting amino acids are replaced with oppositely charged amino acids also leads to a proportion as high as 96% (see, for example, Example 11). Quite surprisingly, the inventors have successfully introduced mutations to further increase the proportion of target immunoglobulin-like molecules in the mixture. For example, Example 17 discloses a method using mutations according to the present invention, wherein the proportion of target bispecific antibodies is increased to such an extent that no dimerization byproducts are detected in the resulting mixture. Unpaired half molecules consisting only of a single heavy chain paired with a common light chain are present in the mixture to a certain extent, but these are the result of unbalanced expression of the heavy chain and can be easily separated from the mixture by size exclusion chromatography. Thus, using such mutations according to the invention, bispecific immunoglobulin-like molecules with a high ratio and substantially no contaminating dimeric by-products can be produced in a single cell, which is particularly suitable for the production of pharmaceutical compositions.

[0065] Therefore, a preferred embodiment of the present invention provides a method for producing a heterodimeric immunoglobulin-like molecule from a single cell, wherein the immunoglobulin-like molecule comprises two CH3 domains capable of forming an interface, the method comprising providing in the cell:

[0066] a. a first nucleic acid molecule encoding a first polypeptide chain containing a CH3 domain,

[0067] b. A second nucleic acid molecule encoding a second CH3 domain-containing polypeptide chain, wherein the first CH3 domain-containing polypeptide chain comprises at least one neutral amino acid residue replaced by a positively charged amino acid residue, and wherein the second CH3 domain-containing polypeptide chain comprises at least one neutral amino acid residue replaced by a negatively charged amino acid residue, the method further comprising the steps of culturing the host cell and allowing the two nucleic acid molecules to be expressed and harvesting the heterodimeric immunoglobulin-like molecule from the culture.

[0068] The method preferably further comprises the step of providing the host cell with a nucleic acid molecule encoding a common light chain, which has the advantages as outlined herein before.

[0069] The amino acid at position 366 of one CH3 domain and the amino acid at position 351 of the second CH3 domain have been reported to be a pair of contact residues at the CH3-CH3 interface, which means that in the resulting three-dimensional conformation of the immunoglobulin-like molecule, they are located close enough to each other to be able to interact with each other. Therefore, the first CH3 domain will preferentially pair with the second CH3 domain.

[0070] In one embodiment, the threonine (T) at position 366 of the first CH3 domain is replaced by a first charged amino acid, and the leucine (L) at position 351 of the second CH3 domain is replaced by a second charged amino acid, wherein the first and second charged amino acids are oppositely charged. If the first CH3 domain-containing polypeptide carrying the charged residue at position 366 also comprises a variable domain with specificity for antigen A, and if the second CH3 domain-containing polypeptide carrying the oppositely charged residue at position 351 also comprises a variable domain with specificity for antigen B, then predominantly bispecific immunoglobulin-like molecules with AB specificity will be formed. Therefore, a method according to the present invention is also provided, wherein the means for preferentially pairing the first CH3 domain-containing polypeptide with the second CH3 domain-containing polypeptide or the means for preferentially pairing the third CH3 domain-containing polypeptide with the fourth CH3 domain-containing polypeptide is replacing the threonine at position 366 of the first or third CH3 domain with a first charged amino acid and replacing the leucine at position 351 of the second or fourth CH3 domain with a second charged amino acid, wherein the first and second charged amino acids have opposite charges.

[0071] A preferred combination of mutations according to the invention is that the Threonine (T) at position 366 of the first CH3 domain-containing polypeptide, which polypeptide also comprises a variable domain (e.g. with specificity A), is replaced by Lysine (K), and the Leucine (L) at position 351 of the second CH3 domain-containing polypeptide, which polypeptide also comprises a variable domain (e.g. with specificity B), is replaced by Aspartic Acid (D). This is denoted as the T366K / L351'D pairing mutation. As explained previously, the amino acid at position 366 of one CH3 domain and the amino acid at position 351 of the second CH3 domain have been reported to be a pair of contact residues at the CH3-CH3 interface. The Lysine introduced at position 366 and the Aspartic Acid introduced at position 351 have opposite charges, making these amino acids electrostatically attracted to each other. Thus, the first CH3 domain will preferentially attract the second CH3 domain and will primarily form immunoglobulin-like molecules comprising a first CH3 domain with a lysine at position 366, which is paired with a second CH3 domain with an aspartic acid at position 351. If the first CH3 domain-containing polypeptide has antigen A specificity, and if the second CH3 domain-containing polypeptide has antigen B specificity, primarily bispecific immunoglobulin-like molecules with "AB" specificity will be formed. Note that in some embodiments, the specificity of the variable domains of both the first and second CH3 domain-containing polypeptide chains may be the same, which will result in the formation of monospecific immunoglobulin-like molecules (e.g. with "AA" specificity). As mentioned above, one of the advantages of the mutations according to the present invention is the fact that new interactions are generated between newly introduced charged amino acid pairs, rather than replacing existing charged amino acid interactions. This has not been previously disclosed or suggested. Therefore, one aspect of the present invention provides a method according to the present invention for producing at least two different immunoglobulin-like molecules from a single host cell, wherein the first CH3 domain-containing polypeptide chain comprises the amino acid substitution T366K and the second CH3 domain-containing polypeptide chain comprises the amino acid substitution L351D. One embodiment provides a method for producing heterodimeric immunoglobulin-like molecules from a single cell, wherein the immunoglobulin-like molecule comprises two CH3 domains capable of forming an interface, the method comprising providing in the cell:

[0072] - a first nucleic acid molecule encoding a first CH3 domain-containing polypeptide chain, and

[0073] - a second nucleic acid molecule encoding a second CH3 domain-containing polypeptide chain, wherein the first CH3 domain-containing polypeptide chain comprises the amino acid substitution T366K, and wherein the second CH3 domain-containing polypeptide chain comprises the amino acid substitution L351D, the method further comprising the steps of culturing the host cell and allowing the two nucleic acid molecules to be expressed and harvesting the heterodimeric immunoglobulin-like molecule from the culture.

[0074] Using the above-mentioned amino acid substitutions according to the present invention, it becomes possible to produce heterodimeric immunoglobulin-like molecules from a single cell, such that the presence of contaminating homodimers is less than 5%, preferably less than 2%, more preferably less than 1%, or most preferably, whereby contaminating homodimers are substantially absent. Therefore, one embodiment provides a method for producing heterodimeric immunoglobulin-like molecules from a single cell, wherein the immunoglobulin-like molecule comprises two CH3 domains capable of forming an interface, and wherein the presence of contaminating homodimers is less than 5%, preferably less than 2%, more preferably less than 1%, and most preferably contaminating homodimers are substantially absent, the method comprising providing in the cell:

[0075] - a first nucleic acid molecule encoding a first CH3 domain-containing polypeptide chain, and

[0076] - a second nucleic acid molecule encoding a second CH3 domain-containing polypeptide chain, wherein the first CH3 domain-containing polypeptide chain comprises the amino acid substitution T366K, and wherein the second CH3 domain-containing polypeptide chain comprises the amino acid substitution L351D, the method further comprising the steps of culturing the host cell and allowing the two nucleic acid molecules to be expressed and harvesting the heterodimeric immunoglobulin-like molecule from the culture.

[0077] Preferably, a method according to the invention for producing at least two different immunoglobulin-like molecules, or a method according to the invention for producing a heterodimeric immunoglobulin-like molecule is provided, wherein the first CH3 domain-containing polypeptide chain further comprises the amino acid substitution L351K. More preferably, the second CH3 domain-containing polypeptide chain further comprises an amino acid substitution selected from the group consisting of Y349E, Y349D and L368E. Most preferably, the second CH3 domain-containing polypeptide chain further comprises the amino acid substitution L368E.

[0078] Therefore, in a preferred embodiment, the above-mentioned T366K / L351'D mutation according to the invention is further combined with a substitution of glutamic acid (E) for leucine (L) at position 368 of the second CH3 domain. For example, this is represented as a T366K / L351'D, L368'E mutation (but alternative representations are also possible, such as T336K / L351D-L368E or T366K / L351D, L368E or T366K-L351D, L368E). As shown in Example 17, the introduction of such mutations according to the invention into a first CH3 domain-containing polypeptide having antigen A specificity and a second CH3 domain-containing polypeptide having antigen B specificity results in a particularly good ratio of bispecific immunoglobulin-like molecules having dual AB specificity. With this mutation pairing, it is even possible to obtain bispecific antibodies without forming any detectable amount of homodimers. Thus, a particularly preferred embodiment provides a method for producing a heterodimeric immunoglobulin-like molecule from a single cell, wherein the immunoglobulin-like molecule comprises two CH3 domains capable of forming an interface and wherein the presence of contaminating homodimers is less than 5%, preferably less than 2%, more preferably less than 1%, and most preferably contaminating homodimers are substantially absent, the method comprising providing in the cell:

[0079] - a first nucleic acid molecule encoding a first CH3 domain-containing polypeptide chain, and

[0080] - a second nucleic acid molecule encoding a second CH3 domain-containing polypeptide chain, wherein the first CH3 domain-containing polypeptide chain comprises the amino acid substitution T366K, and wherein the second CH3 domain-containing polypeptide chain comprises the amino acid substitutions L351D and L368E, the method further comprising the steps of culturing the host cell and allowing the two nucleic acid molecules to be expressed and harvesting the heterodimeric immunoglobulin-like molecule from the culture.

[0081] In another preferred embodiment, the threonine (T) at position 366 of the first CH3 domain is replaced by lysine (K), the leucine (L) at position 351 of the second CH3 domain is replaced by aspartic acid (D) and the tyrosine (Y) at position 349 of the second CH3 domain is replaced by glutamic acid (E). This is, for example, expressed as a T366K / L351'D, Y349'E mutation, but other ways of expressing these mutations may include, for example, T366K-L351D:Y349E or T366K / L351D, Y349E or only T366K / L351DY349E. Residue Y349 is an adjacent residue to residue 351 and may contribute to dimer interactions. According to computer simulation data, Y349E increases the stability of heterodimers (lower computer simulation scores) and the destabilization of homodimers (higher computer simulation scores) and glutamic acid (E) at position 349 is more favorable than aspartic acid (D). Therefore, introducing a second amino acid substitution into a second CH3 domain-containing polypeptide already comprising an amino acid substitution at position 351 further facilitates heterodimerization.

[0082] Thus, a particularly preferred embodiment provides a method for producing a heterodimeric immunoglobulin-like molecule from a single cell, wherein the immunoglobulin-like molecule comprises two CH3 domains capable of forming an interface, and wherein contaminating homodimers are less than 5%, more preferably less than 2%, even more preferably less than 1%, and most preferably substantially absent, the method comprising providing in the cell:

[0083] - a first nucleic acid molecule encoding a first CH3 domain-containing polypeptide chain, and

[0084] - a second nucleic acid molecule encoding a second CH3 domain-containing polypeptide chain, wherein the first CH3 domain-containing polypeptide chain comprises the amino acid substitution T366K, and wherein the second CH3 domain-containing polypeptide chain comprises the amino acid substitutions L351D and Y349E, the method further comprising the steps of culturing the host cell and allowing the two nucleic acid molecules to be expressed and harvesting the heterodimeric immunoglobulin-like molecule from the culture.

[0085] In another preferred embodiment, the threonine (T) at position 366 of the first CH3 domain is replaced by lysine (K), the leucine (L) at position 351 of the second CH3 domain is replaced by aspartic acid (D), the tyrosine (Y) at position 349 of the second CH3 domain is replaced by glutamic acid (E), and the leucine (L) at position 368 of the second CH3 domain is replaced by glutamic acid (E). This is expressed as T366K / L351'D, Y349'E, L368'E mutations. These two residues Y349 and L368 are residues that may contribute to dimer interaction. According to computer simulation data, Y349E and L368E increase the stability of the heterodimer (lower computer simulation score) and the instability of the BB dimer (higher computer simulation score) and the glutamic acid (E) at positions 349 and 368 is more favorable than aspartic acid (D). Thus, heterodimerization is further favored by introducing the second and third amino acid substitutions in the B chain, which already contains the amino acid substitution at position 351. Thus, a particularly preferred embodiment provides a method for producing a heterodimeric immunoglobulin-like molecule from a single cell, wherein the immunoglobulin-like molecule comprises two CH3 domains capable of forming an interface, and wherein contaminating homodimers are less than 5%, more preferably less than 2%, even more preferably less than 1%, and most preferably substantially absent, the method comprising providing in the cell:

[0086] - a first nucleic acid molecule encoding a first CH3 domain-containing polypeptide chain, and

[0087] - a second nucleic acid molecule encoding a second CH3 domain-containing polypeptide chain, wherein the first CH3 domain-containing polypeptide chain comprises the amino acid substitution T366K, and wherein the second CH3 domain-containing polypeptide chain comprises the amino acid substitutions L351D and Y349E and L368E, the method further comprising the steps of culturing the host cell and allowing the at least two nucleic acid molecules to be expressed and harvesting the heterodimeric immunoglobulin-like molecule from the culture.

[0088] In another preferred embodiment, the threonine (T) at position 366 of the first CH3 domain is replaced by lysine (K), the leucine (L) at position 351 of the first CH3 domain is replaced by lysine (K), the leucine (L) at position 351 of the second CH3 domain is replaced by aspartic acid (D), and the leucine (L) at position 368 of the second CH3 domain is replaced by glutamic acid (E). This is represented by T366K, L351K / L351'D, L368'E mutations. This mutation also enhances the proportion of the target (bispecific) antibody, as shown in the examples. Similarly, the use of these mutations also enables bispecific antibodies to be obtained without forming any detectable amount of homodimers. Therefore, a method for producing heterodimeric immunoglobulin-like molecules from a single cell is also provided, wherein the immunoglobulin-like molecule comprises two CH3 domains capable of forming an interface, and wherein the contaminating homodimers are less than 5%, preferably less than 2%, more preferably less than 1%, and most preferably substantially absent, the method comprising providing in the cell:

[0089] - a first nucleic acid molecule encoding a first CH3 domain-containing polypeptide chain, and

[0090] - a second nucleic acid molecule encoding a second CH3 domain-containing polypeptide chain, wherein the first CH3 domain-containing polypeptide chain comprises amino acid substitutions T366K and L351K, and wherein the second CH3 domain-containing polypeptide chain comprises amino acid substitutions L351D and L368E, the method further comprising the steps of culturing the host cell and allowing the two nucleic acid molecules to be expressed and harvesting the heterodimeric immunoglobulin-like molecule from the culture.

[0091] In another preferred embodiment, the threonine (T) at position 366 of the first CH3 domain is replaced by lysine (K), the leucine (L) at position 351 of the first CH3 domain is replaced by lysine (K), the leucine (L) at position 351 of the second CH3 domain is replaced by aspartic acid (D), the tyrosine (Y) at position 349 of the second CH3 domain is replaced by aspartic acid (D), and the arginine (R) at position 355 of the second CH3 domain is replaced by aspartic acid (D). This is represented by T366K, L351K / L351'D, Y349'D, R355'D mutations. The T366K-L351K / L351'D-Y349'D pairing can be further improved by the R355'D mutation of the B chain, which results in a higher BB computer simulation score, and the AB computer simulation score is also slightly improved. Therefore, there is further provided a method for producing a heterodimeric immunoglobulin-like molecule from a single cell, wherein the immunoglobulin-like molecule comprises two CH3 domains capable of forming an interface, and wherein contaminating homodimers are less than 5%, more preferably less than 2%, even more preferably less than 1%, and most preferably substantially absent, the method comprising providing in the cell:

[0092] - a first nucleic acid molecule encoding a first CH3 domain-containing polypeptide chain, and

[0093] - a second nucleic acid molecule encoding a second CH3 domain-containing polypeptide chain, wherein the first CH3 domain-containing polypeptide chain comprises amino acid substitutions T366K and L351K, and wherein the second CH3 domain-containing polypeptide chain comprises amino acid substitutions L351D and Y349D and R355D, the method further comprising the steps of culturing the host cell and allowing the two nucleic acid molecules to be expressed and harvesting the heterodimeric immunoglobulin-like molecule from the culture.

[0094] Table B provides an overview of mutations that can be introduced into the CH3 domain as a preferred means for preferential pairing to generate heterodimers or homodimers.

[0095] Table B:

[0096] Amino acid substitutions in CH3 Constructs# Prioritize pairing with -(wild type) - wild type E356K, D399K 1 Construct 2 or 3 K392D, K409D 2 Construct 1 K392D, K409D, K439D 3 Construct 1 K392D, D399K, K409D 4 Construct 4 E356K, E357K, K439D, K370D 5 Construct 5 T366W 6 Construct 7 T366S, L368A, Y407V 7 Construct 6 T366K 43 Constructs 63, 69, 70, 71, 73 L351D 63 Construct 43, 68 T366K, L351K 68 Constructs 63, 69, 70, 71, 72, 75 L351D, L368E 69 Construct 43, 68 L351E,Y349E 70 Construct 43, 68 L351D,Y349E 71 Construct 43, 68 L351D, R355D 72 Construct 43, 68 L351D, Y349E, L368E 73 Construct 43 L351D, Y349D, R355D 75 Construct 68

[0097] Therefore, also provided herein is a method according to the invention for producing at least two different immunoglobulin-like molecules, or a method according to the invention for producing a heterodimeric immunoglobulin-like molecule, wherein said means for preferentially pairing said first CH3 domain-containing polypeptide with said second CH3 domain-containing polypeptide and / or said means for preferentially pairing said third CH3 domain-containing polypeptide with said fourth CH3 domain-containing polypeptide comprises at least one combination of the mutations shown in Table B. Preferably, said means for preferentially pairing said first CH3 domain-containing polypeptide with said second CH3 domain-containing polypeptide and said means for preferentially pairing said third CH3 domain-containing polypeptide with said fourth CH3 domain-containing polypeptide comprises at least two combinations of the mutations shown in Table B.

[0098] The present invention also provides new combinations of CH3 mutations, with which it is possible to produce a mixture of at least two monospecific immunoglobulin-like molecules in a single cell, wherein the contaminating bispecific immunoglobulin-like molecules are less than 5%, preferably more than 2%, even more preferably less than 1%, and most preferably even substantially absent. Therefore, these mutations according to the present invention are particularly suitable for producing mixtures of monospecific antibodies, which is advantageous in the following situations, for example, when it is desired that two identical target molecules have a high level of cross-linking, when the density of antibodies on the target cells is required to be high enough to recruit certain effector functions (such as complement-mediated lysis of tumor cells), or when the two targets are so far away from each other that they cannot be bound by a single bispecific antibody, or in order to simplify the approval procedures of regulatory authorities. In these cases, it is generally desirable to optimize the production platform of such monospecific antibodies. As shown in Example 10, the present invention provides the recognition that when lysine (K) at position 392 of the first CH3 domain-containing polypeptide (e.g., with specificity A) is replaced by aspartic acid (D), and when aspartic acid (D) at position 399 of the first CH3 domain-containing polypeptide is replaced by lysine (K), and when lysine (K) at position 409 of the first CH3 domain-containing polypeptide is replaced by aspartic acid (D), it is possible to produce a mixture of at least two different monospecific immunoglobulin-like molecules in a single cell, including a monospecific immunoglobulin-like molecule with AA specificity, wherein the formation of bispecific side products (bispecific immunoglobulin-like molecules) is reduced to less than 5%, or even less than 3% or even substantially undetectable. Therefore, the combination of the above mutations (referred to herein as K392D, D399K, K409D) is particularly preferred for producing a mixture of monospecific immunoglobulin-like molecules. Those skilled in the art will appreciate that its functional variants, i.e. K392E, D399R, K409E, can lead to similar effects. In addition, double mutants (including D399K and K409D substitutions) or other functional variants such as K392D and K409D, D399R and K409E, etc. may also lead to similar effects.

[0099] The same applies to the combination of mutations wherein Glutamic acid (E) at position 356 of the first CH3 domain-containing polypeptide is replaced by Lysine (K), and wherein Glutamic acid (E) at position 357 of the first CH3 domain-containing polypeptide is replaced by Lysine (K), and wherein Lysine (K) at position 439 of the first CH3 domain-containing polypeptide is replaced by Aspartic acid (D), and wherein Lysine (K) at position 370 of the first CH3 domain-containing polypeptide is replaced by Aspartic acid (D). The combination of mutations (referred to herein as E356K, E357K, K439D, K370D) is also particularly preferred for generating a mixture of monospecific immunoglobulin-like molecules. A person skilled in the art will appreciate that their functional variants, i.e. E356R, E357R, K439E, K370E, can lead to similar effects. In addition, triple or double mutants (including E356K and K439D and E357K and K370D substitutions) or other functional variants may also lead to similar effects. Therefore, another embodiment provides a method for producing at least two different monospecific immunoglobulin-like molecules from a single host cell, wherein the two immunoglobulin-like molecules each comprise two CH3 domains capable of forming an interface, the method comprising providing in the cell:

[0100] a) a first nucleic acid molecule encoding a first CH3 domain-containing polypeptide chain having A specificity,

[0101] b) a second nucleic acid molecule encoding a second CH3 domain-containing polypeptide chain having B specificity,

[0102] Wherein, the first CH3 domain-containing polypeptide chain comprises K392D, D399K, K409D mutations and the second CH3 domain-containing polypeptide chain comprises a wild-type CH3 domain or comprises E356K, E357K, K439D, K370D mutations, and the method further comprises the steps of culturing the host cell and allowing the nucleic acid molecule to be expressed and obtaining the at least two different immunoglobulin-like molecules from the culture.

[0103] An alternative embodiment provides a method for producing at least two different monospecific immunoglobulin-like molecules from a single host cell, wherein each of the two immunoglobulin-like molecules comprises two CH3 domains capable of forming an interface, the method comprising providing in the cell:

[0104] a) a first nucleic acid molecule encoding a first CH3 domain-containing polypeptide chain having A specificity,

[0105] b) a second nucleic acid molecule encoding a second CH3 domain-containing polypeptide chain having B specificity,

[0106] Wherein, the first CH3 domain-containing polypeptide chain comprises a wild-type CH3 domain or comprises K392D, D399K, K409D mutations, and the second CH3 domain-containing polypeptide chain comprises E356K, E357K, K439D, K370D mutations, and the method further comprises the steps of culturing the host cell and allowing the nucleic acid molecule to be expressed and harvesting the at least two immunoglobulin-like molecules from the culture.

[0107] As shown in Example 10, two monospecific immunoglobulin-like molecules can be produced in a single cell, wherein the formation of bispecific immunoglobulin-like molecules is essentially undetectable. A person skilled in the art can select a third nucleic acid molecule encoding a wild-type or modified CH3 domain-containing polypeptide chain to provide to the host cell, thereby producing a mixture of three monospecific antibodies, and so on.

[0108] In one aspect of the invention, a method according to the invention is provided for producing at least two different immunoglobulin-like molecules or for producing heterodimeric immunoglobulin-like molecules, wherein the polypeptide chains containing the CH3 domain also each comprise a variable region that recognizes a different target epitope, wherein the target epitope is located on the same molecule. This generally enables more effective antagonism of the (biological) function of the target molecule compared to the case where only one epitope is targeted. For example, a heterodimeric immunoglobulin-like molecule can simultaneously bind to two epitopes present on, for example, a growth factor receptor or soluble molecule that is critical for the proliferation of tumor cells, thereby effectively preventing multiple independent signal transduction pathways that lead to uncontrolled cell proliferation, and any combination of at least two immunoglobulin-like molecules can simultaneously bind to 2, or even 3 or 4 epitopes present on such a growth factor receptor or soluble molecule.

[0109] In a preferred embodiment, the target molecule is a soluble molecule. In another preferred embodiment, the target molecule is a membrane bound molecule.

[0110] In another aspect of the present invention, a method according to the present invention is provided for producing at least two different immunoglobulin-like molecules or for producing heterodimeric immunoglobulin-like molecules, wherein the polypeptide chains containing the CH3 domain each also include a variable region that recognizes a target epitope, wherein the target epitope is located on different molecules. In this case, each of the different target molecules can be a soluble molecule or a membrane-bound molecule. In one embodiment, the different target molecules are all soluble molecules. Alternatively, one target molecule is a soluble molecule and the second target molecule is a membrane-bound molecule. In another alternative, both target molecules are membrane-bound molecules. In one embodiment, the different target molecules are expressed on the same cell, while in other embodiments, the different target molecules are expressed on different cells. As a non-limiting example, any heterodimeric immunoglobulin-like molecule or any combination of at least two immunoglobulin-like molecules can be suitable for simultaneously blocking multiple membrane-bound receptors, neutralizing multiple water-soluble molecules (e.g., cytokines or growth factors for tumor cells), or neutralizing different viral serotypes or strains.

[0111] A preferred embodiment provides a method according to the present invention for producing at least two different immunoglobulin-like molecules or for producing heterodimeric immunoglobulin-like molecules, wherein at least one of the target epitopes is located on tumor cells. Alternatively or additionally, at least one of the target epitopes is located on the surface of effector cells. This is suitable for, for example, recruiting T cells or NK cells for tumor cell killing. For example, at least one immunoglobulin-like molecule produced according to the method of the present invention, by specifically binding to a target molecule located on an immune effector cell, can recruit immune effector cells, preferably human immune effector cells. In another embodiment, once an immunoglobulin-like molecule is bound to a target molecule, the immune effector cell is activated. Effector mechanism recruitment can, for example, encompass: by administering an immunoglobulin-like molecule produced according to the method of the present invention that can bind to a cytotoxic trigger molecule (such as a T cell receptor or Fcγ receptor), the immunomodulatory cytotoxicity is redirected, thereby activating downstream immune effector pathways. As used herein, the term "immune effector cell" or "effector cell" refers to a cell in the natural cell pool of the mammalian immune system, which can be activated to affect the viability of a target cell. Immune effector cells include cells of lymphoid system, such as natural killer (NK) cells, T cells (including cytotoxic T cells or B cells), and cells of myeloid system can also be considered as immune effector cells, such as monocytes or macrophages, dendritic cells and neutrophils. Therefore, the effector cells are preferably NK cells, T cells, B cells, monocytes, macrophages, dendritic cells or neutrophils.

[0112] The target antigen presented by immune effector cells can include CD3, CD16, CD25, CD28, CD64, CD89, NKG2D and NKp46. Therefore, a method for producing at least two different immunoglobulin-like molecules or for producing heterodimeric immunoglobulin-like molecules according to the present invention is also provided, wherein the target epitope is located on CD3, CD16, CD25, CD28, CD64, CD89, NKG2D or NKp46 molecules.

[0113] Viability of a target cell may include the ability of the cell to survive, proliferate, and / or interact with other cells.

[0114] Therefore, in one aspect, the present invention provides a method for producing a heterodimeric immunoglobulin-like molecule according to the present invention, wherein the polypeptide chains containing the CH3 domain also each comprise a variable region that recognizes a target epitope. In one embodiment, the two variable regions of the polypeptide chain containing the CH3 domain each recognize the same target epitope, but have different affinities. In another embodiment, the two variable regions of the polypeptide chain containing the CH3 domain each recognize a different target epitope. In another embodiment, the different target epitopes are located on the same target molecule, which may be a membrane-bound molecule or a soluble molecule. In another embodiment, the different target epitopes are located on different target molecules, which may be expressed on the same cell or on different cells. Alternatively, the different target molecules may be soluble molecules, or one target molecule may be a soluble molecule and the second target molecule is a membrane-bound molecule. In a preferred embodiment, at least one target molecule of the heterodimeric immunoglobulin-like molecule is located on a tumor cell. In another preferred embodiment, at least one target molecule of the heterodimeric immunoglobulin-like molecule is located on an effector cell (i.e., a NK cell, a T cell, a B cell, a monocyte, a macrophage, a dendritic cell or a neutrophil, and the target epitope may be located on a CD3, CD16, CD25, CD28, CD64, CD89, NKG2D or NKp46 molecule).

[0115] In a preferred embodiment, a method for producing at least two different immunoglobulin-like molecules or for producing heterodimeric immunoglobulin-like molecules according to the present invention is provided, wherein the at least two different immunoglobulin-like molecules are antibodies, most preferably IgG isotype antibodies, even more preferably IgG1 isotype as described above.

[0116] Also provided is an immunoglobulin-like molecule, a heterodimer immunoglobulin-like molecule, or a mixture of at least two immunoglobulin-like molecules that can be obtained by the method according to the present invention. The mixture of the (heterodimer) immunoglobulin-like molecule or immunoglobulin-like molecule preferably comprises at least one CH3 mutation as described in Table B. Therefore, also provided is a (heterodimer) immunoglobulin-like molecule or a mixture of at least two immunoglobulin-like molecules comprising at least one mutation as described in Table B, and a pharmaceutical composition comprising at least one immunoglobulin-like molecule or a mixture of at least two immunoglobulin-like molecules according to the present invention. In one embodiment, the immunoglobulin-like molecule is a bispecific immunoglobulin-like molecule, such as a bispecific antibody. In another embodiment, the immunoglobulin-like molecule is a monospecific immunoglobulin-like molecule, such as a monospecific antibody. A preferred embodiment provides a mixture of at least two different immunoglobulin-like molecules that can be obtained by the method according to the present invention, wherein the at least two different immunoglobulin-like molecules are combined with different epitopes on the same antigen and / or are combined with different epitopes on different antigens. Also provided are heterodimeric immunoglobulin-like molecules obtainable according to the method of the present invention, wherein the heterodimeric immunoglobulin-like molecules bind to different epitopes on the same antigen and / or bind to different epitopes on different antigens. The advantages and preferred uses of such mixtures and antibodies are described herein before. The present invention also provides a mixture of at least two different immunoglobulin-like molecules obtainable according to the method of the present invention, wherein the at least two different immunoglobulin-like molecules include at least one heterodimeric immunoglobulin-like molecule. In one embodiment, both of the at least two different immunoglobulin-like molecules are heterodimeric immunoglobulin-like molecules. Another preferred embodiment provides a heterodimeric antibody comprising 2 CH3 domains, wherein one of the two CH3 domains comprises amino acid replacements L351D and L368E, and wherein the other of the two CH3 domains comprises amino acid replacements T366K and L351K. As explained above, these amino acid replacements are preferred means for preferentially pairing the two CH3 domains. The amino acid substitutions L351D and L368E in one of the two CH3 domains and the amino acid substitutions T366K and L351K in the other of the two CH3 domains are collectively referred to as 'DEKK mutation combination', 'DEKK variant', 'DEKK pair', 'DEKK engineered CH3 domain', 'DEKK' or alternative names using reference to DEKK. The CH3 domain carrying the amino acid substitutions L351D and L368E is also referred to as the 'DE side' and the CH3 domain carrying the amino acid substitutions T366K and L351K is also referred to as the 'KK-side'.

[0117] Also provided is a pharmaceutical composition comprising a (heterodimer) immunoglobulin-like molecule or a mixture of at least two immunoglobulin-like molecules obtainable according to the method of the present invention. The (heterodimer) immunoglobulin-like molecule or the at least two immunoglobulin-like molecules according to the present invention are preferably antibodies. The pharmaceutical composition may comprise the (heterodimer) immunoglobulin-like molecule, a mixture of monospecific or bispecific immunoglobulin-like molecules, or a combination of monospecific and bispecific immunoglobulin-like molecules. In addition, the pharmaceutical composition according to the present invention comprises a pharmaceutically acceptable carrier. As used herein, such a "pharmaceutically acceptable carrier" includes any and all solvents, salts, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents and other physiologically compatible substances. Depending on the route of administration (e.g., intravenous, subcutaneous, intra-articular, etc.), the immunoglobulin-like molecule can be coated in a material to protect the immunoglobulin-like molecule from the effects of acid and other natural conditions that may inactivate the immunoglobulin-like molecule. In one aspect, a pharmaceutical composition comprising a mixture of at least two immunoglobulin-like molecules obtainable by any method according to the present invention is provided, wherein the at least two different immunoglobulin-like molecules are produced by a recombinant host cell according to the present invention. In addition, a pharmaceutical composition comprising a heterodimeric immunoglobulin-like molecule obtainable by any method according to the present invention is provided, wherein the heterodimeric immunoglobulin-like molecule is produced by a recombinant host cell according to the present invention.

[0118] Also provided herein are nucleic acid molecules encoding a CH3 domain-containing polypeptide chain containing at least one mutation shown in Table B, and recombinant host cells comprising at least one nucleic acid molecule encoding a CH3 domain-containing polypeptide chain containing at least one mutation shown in Table B.

[0119] The present invention is further illustrated by the following examples. These examples are not intended to limit the present invention in any way, but are merely used to illustrate the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0120] Figure 1 : A) Schematic diagram of construct vector MV1057. The filled area is the area where the antibody VH region is cloned in. B) Schematic diagram of phage display vector MV1043.

[0121] Figure 2 : Amino acid sequence of wild-type IgG1 Fc as present in the construct vector MV1057 (EU numbering scheme applied).

[0122] Figure 3 : Nucleotide and amino acid sequences of the VH region used for cloning into various constructs.

[0123] Figure 4 : Mass spectrometry data of transfection A, G and H.

[0124] Figure 5 : Mass spectrometry data of transfected M and U.

[0125] Figure 6 : Mass spectrometry data of transfected O.

[0126] Figure 7 : Homodimerization is prevented by replacing neutral amino acids with charged amino acids.

[0127] Figure 8 : Native MS spectrum of transfected sample ZO (T366K / L351'D) (A) and convoluted MS spectrum of transfected sample ZO (T366K / L351'D). The second / main peak represents the bispecific molecule (B).

[0128] Fig. 9 : Experimental validation of HADDOCK scores of mutation pairs.

[0129] Fig.10 : Cartoon diagram of CH3-CH3 interface interaction; A) K409D: K392D / D399'K: E356'K, B) D399K: E356K / D399'K: E356'K, C) K409D: K392D / K409'D: K392'D.

[0130] Fig.11 : HADDOCK scores of various 366 / 351' charge mutants.

[0131] Fig.12 : Cartoon diagram of CH3-CH3 interface interaction; A) L351D / L351'D, B) L351D:S354A:R355D / L351'D:S354'A:R355'D.

[0132] Fig.13 : HADDOCK scores for additional charge mutations near position L351.

[0133] Fig.14 : HADDOCK scores for additional charge mutations near position T366 in chain A and position L351 in chain B.

[0134] Fig.15 : Cartoon illustration of CH3-CH3 interface interaction.

[0135] Fig.16 : HADDOCK scores of mutations near T366 / L351.

[0136] Fig.17 : HADDOCK scores of additional variants near T366 / L351.

[0137] Fig.18 : Examples of nMS spectra of bispecific immunoglobulins obtained after co-expression of construct T366K, L351K with construct L351D (left-hand panel) or L351D, Y349E (right-hand panel), zooming in on the singly charged state of the intact IgG (half bodies not shown).

[0138] Fig.19A : The native MS results show the relative abundance of AA, AB, BB, A, and B (all species total 100%); Fig.19B : Same as above, but now without AB, in order to have a better understanding of the undesirable categories AA, BB, A and B.

[0139] Fig. 20 : Thermostability assay results. Squares: wild type; triangles: charge-reversed pair E356K:D399K / K392D:K409D; circles: mutant CH3 combinations shown in each figure above.

[0140] Fig.21 : Results of 10× freeze-thaw experiments. 1122 = 1st parent antibody BB; 1337 = 2nd parent antibody AA; Wild type = AA, AB, BB; CR = bispecific for charge-reversed pair E356K:D399K / K392D:K409D; 3-6 and 9-12 = bispecific molecules from the combination of 3-6 and 9-12 in Table 15.

[0141] Fig. 22 : Serum stability results, measured by ELISA using fibrinogen as coating antigen. A) ELISA data of IgG samples diluted to 0.5 μg / ml; B) ELISA data of IgG samples diluted to 0.05 μg / ml; Results are normalized to the time point of T=0 days (100%). 1337=second parent antibody AA; Wild type=AA, AB, BB; CR=charge-reversed bispecific for E356K:D399K / K392D:K409D; 3-6 and 9-12=bispecific molecules from the combination of 3-6 and 9-12 from Table 15.

[0142] Fig.23: nMS results of ratio experiments with transfection ratios ranging from 1:5 to 5:1. A) DEKK mutation combination with specificity "A" on the DE side and specificity "B" on the KK side; B) DEKK mutation combination with specificity "C" on the DE side and specificity "B" on the KK side; C) Charge reversal combination of mutations with specificity "A" on the E356K:D399K side and specificity "B" on the K392D:K409D side.

[0143] Fig.24 : nMS results for transfections #1-11 from Table 20.

[0144] Fig.25 : HADDOCK scores of dimers with different CH3 engineered vectors. Grey bars: expected species AB and CD; black bars: undesired species AA, BB, CC, DD, AC, BC, AD, BD.

[0145] Fig.26 : SDS-PAGE of transfections #1-11 from Table 20. Control samples DE / KK, DE / DE, and KK / KK were also included.

[0146] Figure 27A-27C : nMS transfected with #9 (A, B) and transfected with #11 (C).

[0147] Fig.28 : nMS of gel filtration samples 1516:1516 (A), 1337:1337 (B) and 1516:1337 (C).

[0148] Fig.29 : Serum levels of samples of the DEKK engineered antibody and its two parental antibodies (pK study). DETAILED DESCRIPTION

[0149] Example

[0150] Example 1: Amino acid substitution to generate a variety of different CH3 domains

[0151] Several amino acid substitutions known to promote heterodimer formation as well as several alternative amino acid substitutions that have not been previously reported or tested but were selected to promote homodimer formation were introduced into the construct vector (construct vector MV1057; Figure 1 A) to obtain a wide variety of immunoglobulin-like molecules with different CH3 domains, so that the pairing of CH3 domains (including immunoglobulin-like molecules) is preferentially promoted or inhibited. Figure 2As shown, construct vector MV1057 comprises the nucleic acid sequence encoding normal wild-type IgG1 Fc part. Table 1 lists the amino acid replacement introduced into this wild-type Fc, and a series of seven constructs are produced. All constructs are produced at Geneart. As constructs 6 and 7 (WO98 / 50431), constructs 1, 2 and 3 have been previously described, or their substitutes drive heterodimerization (EP01870459, WO2009 / 089004). Constructs 4 and 5 are new and are designed to promote homodimerization.

[0152] Table 1

[0153]

[0154] Example 2: Cloning of VH into constructs containing CH3 mutations

[0155] Several antibody VH regions with known specificity and known to be able to pair with human IGKV1-39 light chains were used for cloning into these constructs. As previously described, all CH3 variants can be used to associate with other antibody domains to produce bispecific or monospecific full-length antibodies. The antibody specificity defined by the VH / VL combination will not affect the heavy chain dimerization behavior driven by the CH3 domain. Throughout the study, a model VH / VL combination was used in which all VLs were based on germline human IGKV1-39 and the VH was changed. Figure 3 The full sequences and specificities of the antibody VH regions used throughout the study are provided. The MF codes refer to the internal Merus names of the different VHs, for example, VH MF1337 is specific for tetanus toxoid, MF1025 is specific for porcine thyroglobulin, and MF1122 is specific for bovine fibrinogen. The phage display vector MV1043 ( Figure 1 The VH region present in B) of the vector is removed to release the VH fragment from the vector. The vector MV1057 is digested with Sfil and BstEII according to standard methods (according to the manufacturer's instructions). The fragment and vector are gel purified (Promega / Cat. No. #V3125 / according to the manufacturer's instructions) to separate the cut vector and the VH gene insert. The two are combined by ligation, and the ligation product is then transformed into E. coli DH5α (Invitrogen / Cat. No. #12297-016 / according to the manufacturer's instructions). After overnight screening, single colonies are picked and the vector containing the correct insert is identified by sequencing.

[0156] Example 3: Transfection and expression of whole IgG in HEK293T cells

[0157] According to standard methods, various plasmids encoding recloned VH variants and also encoding the common light chain huIGKV1-39 were transfected in HEK293T cells to allow IgG expression (de Kruif et al. Biotech Bioeng. 2010). After transfection, the expression level of IgG in the supernatant was measured using the ForteBIO Octet-QK system, which is based on bio-layer interferometry (BLI) and enables real-time quantification and kinetic characterization of biomolecular interactions; for details, see www.fortebio.com When expression levels exceeding 5 μg / ml were measured, IgG was purified using protein A affinity purification.

[0158] Example 4: Purification of IgG

[0159] Culture supernatants were purified using protein A columns (GE Healthcare / catalog # 11-0034-95 / according to the manufacturer's instructions), eluted with 0.1 M citrate buffer pH 3.0, and immediately neutralized with an equal volume of 1.0 M Tris-HCL pH 8.0, or directly rebuffered into PBS using a desalting column. Alternatively, IgG can be purified using protein A beads (Sepharose beads CL-4B, GE healthcare catalog # 170780-01).

[0160] Example 5: Antigen-specific ELISA's

[0161] Antigen-specific ELISA was performed to determine the binding activity to the antigen and capture ELISA was performed to demonstrate the binding activity of the bispecific antibody. The complex was detected using a biotinylated secondary antibody (de Kruif et al. Biotech Bioeng. 2010).

[0162] Example 6: SDS-PAGE

[0163] The samples were analyzed by SDS-PAGE ( The purified IgG mixture was analyzed by 4-12% bis-tris gel (Invitrogen / Catalog # NP0323BOX) and stained with Colloidal Blue (PageBlue TM Protein staining solution / Fermentas / Cat. #RO571) was used to stain proteins in the gel.

[0164] Example 7: Enzymatic deglycosylation of IgG1

[0165] Due to the heterogeneity in the glycosylation of IgG, the protein was deglycosylated to produce a single product with different masses suitable for mass spectrometry analysis. One unit of N-glycosidase F (PNGase F; Roche Diagnostics, Mannheim, Germany) was incubated overnight at 37°C for every 10 μg of IgG1. Buffer exchange was performed using a 10 kDa MWCO centrifugal filter column (Millipore) to remove the initial purification buffer (0.1 M citrate buffer pH 3.0 / 1.0 M Tris-HCL pH 8.0) and rebuffered to PBS. A similar buffer exchange step was performed to remove the separated glycan chains and the buffer was exchanged for 150 mM ammonium acetate at pH 7.5. The filter was washed with 200 μL of 150 mM ammonium acetate at pH 7.5 for 12 minutes at 4°C and 11,000 rpm. After washing, 50 μL of deglycosylated IgG was loaded onto the filter and 450 μL of 150 mM ammonium acetate pH 7.5 was added, followed by another round of centrifugation for 12 minutes at 4°C and 11,000 rpm. In total, the centrifugation was repeated 5 times, adding fresh 150 mM ammonium acetate pH 7.5 buffer each time to a total volume of 500 μL. After the final centrifugation step, approximately 25 μL of the remaining buffer-exchanged deglycosylated IgG1 was collected and transferred to a microcentrifuge tube in preparation for mass spectrometry analysis.

[0166] Example 8: Intrinsic mass spectrometry analysis

[0167] Mass spectrometry was used to identify the different IgG species in the purified IgG mixture, and to determine the ratios at which these IgG species exist. In brief, 2 to 3 μl of 1 μM concentration of IgG in ammonium acetate of 150 mM pH 7.5 was added to a gold-plated borosilicate capillary made in-house (using Sutter P-97 puller [Sutter Instruments Co., Novato, CA, USA] and Edwards Scancoat six sputterers (sputter-coater) [Edwards Laboratories, Milpitas, CA, USA), to be analyzed on LCT 1 mass spectrometry (Waters Corp., Milford, MA, USA), and the mass spectrometry was adjusted to have optimal performance in high mass detection (Tahallah et al., RCM 2001). The capillary voltage of 1300 V was used, and the sampling cone voltage was 200 V; however, when a higher resolution "signal-to-noise ratio" was required, these settings were adjusted. The source backing pressure was increased to induce collision cooling to about 7.5 mbar. Proteins were sprayed at a concentration of 1 μM in 5% formic acid for determination of IgG1 under denaturing conditions.

[0168] Example 9: Data processing and quantification

[0169] Utilize MassLynx 4.1 software (Waters Corp., Milford, MA, USA), the spectrum that obtains is processed.Use minimum filtering (smoothing), then concentrate the spectrum.Use each charge state in series to calculate the mass of species.By MassLynx, assign the corresponding intensity of each charge state, and add.This method enables relative quantification of all species in one sample.Alternatively, utilize the area under the curve (AUC) method known in the art to carry out quantification of peak.All analyses are repeated three times to calculate the standard deviation of IgG mass and its relative abundance.

[0170] Example 10: Cocktail of 2 or 3 monospecific antibodies from a single cell

[0171] Several light chains with known specificity and known to bind to the human IGKV1-39 light chain ( Figure 3) The antibody VH region paired with MV1057 was used for recloning into the wild-type construct vector MV1057, or construct 4 or construct 5 in Table 1, thereby obtaining vectors I to III (Table 2). Subsequently, the obtained vectors I, II and III were transfected into cells alone to prove that only complete monospecific antibodies were formed, or transfected into cells in combination with one or two other construct vectors to obtain a mixture of two monospecific or three monospecific antibodies, each of which contained nucleic acid sequences encoding a common human light chain and an Ig heavy chain with different CH3 regions and different VH specificities. Table 3 shows the transfection scheme and results.

[0172] Table 2: VH specificity inserted in different constructs

[0173]

[0174] Table 3: Transfection protocol and results

[0175]

[0176] nd = not performed.

[0177] It was observed that transfection of A, G and H resulted in the formation of only homodimers, and 100% of bivalent monospecific AA, BB or CC ( Figure 4 Although this was expected and previously demonstrated in transfection A, the fact that homodimerization of CH3 engineered Ig heavy chains comprising either the ternary amino acid substitutions of construct 4 (i.e., K392D, D399K, K409D) or the quaternary amino acid substitutions of construct 5 (i.e., E356K, E357K, K439D, K370D) is now reported for the first time (transfections G and H).

[0178] Next, a co-expression experiment of the two vectors was performed in a single cell. Interestingly, transfection M and N showed that the wild-type and CH3-modified Ig heavy chains can be co-expressed in a single cell together with a common light chain, thereby producing a mixture of two monospecific antibodies without the presence of undesirable bispecific antibodies in the mixture, but with as little as 4% to 5% of contaminating "other molecules". "Other molecules" are defined as all molecules that do not have the quality of a complete IgG, including half molecules consisting of a single heavy chain and light chain pair. Importantly, some "others" do not include bispecific products. In transfection M, when equal ratios of vector DNA were transfected, the ratio of AA:BB was close to 1:1. However, transfection N resulted in a ratio of AA:CC of almost 10:1. Therefore, the transfection was repeated with DNA adjusted in ratio (transfection U). In fact, the ratio of 1:5 of vector DNA I:III equalized the ratio of AA:CC antibody products in the mixture, tending to be close to a ratio of 1:1. Thus, transfection of M and U demonstrated that two different substantially pure monospecific antibodies (A and B) can be expressed in a single cell without undesirable byproducts (i.e., without the presence of significant amounts of AC or half molecules A or C). Figure 5 ). The novel CH3 modifications of constructs 4 and 5 are significantly different from the wild-type CH3, so that heterodimerization does not occur between the wild-type and 4 or the wild-type and 5, which is beneficial for the application of large-scale production of monospecific antibody mixtures from a single cell.

[0179] Similar to these results, it is also expected that the transfection of two different CH3 engineered Ig heavy chains (Constructs 4 and 5) only results in a mixture of two different monospecific antibodies, without the presence of other undesirable species. It is inferred that the CH3 modification of Construct 4 is significantly different from that of Construct 5, so that heterodimerization does not occur. In this case, the co-expression of the CH3 engineered heavy chains of Constructs 4 and 5 together with the wild-type CH3 heavy chain in a single cell only results in three monospecific antibodies.

[0180] In fact, it was observed that this is exactly the case, i.e. it was found that a mixture of three pure monospecific antibodies can also be obtained by expressing three different Ig heavy chains (designed to form homodimers rather than heterodimers) together with a common light chain in a single cell, and in the absence of contaminating substances in the mixture (transfection O) ( Figure 6 ). As can be seen in Table 3, with equal ratios of carrier DNA used during transfection O, no 1:1:1 ratio of AA:BB:CC antibodies was obtained. Transfections using altered carrier DNA ratios (1:1:10, transfection V) demonstrated that the ratio of AA:BB:CC in the mixture can be controlled towards the desired ratio.

[0181] In summary, these experiments demonstrate that two or three substantially pure monospecific antibodies can be expressed in a single cell without undesired byproducts, facilitating large-scale production of mixtures of therapeutic monospecific antibodies.

[0182] Example 11: Mixture of two bispecific antibodies from a single cell

[0183] Given that the use of CH3 engineered heavy chains for the generation of a single bispecific antibody has been reported elsewhere, this experiment was designed to investigate whether it is feasible to produce a mixture of two different bispecific antibodies from a single cell.

[0184] A light chain with known specificity and known to bind to human IGKV1-39 ( Figure 3 ) paired antibody VH region is used for recloning into a vector containing constructs 1 to 3 or 6 to 7 in Table 1, thereby obtaining vectors IV to X (Table 4). Subsequently, vectors IV to X are transfected into cells alone to demonstrate that the formation of complete monospecific antibodies is restricted, or are transfected into cells in combination with another construct vector to obtain bispecific antibodies or a mixture of two bispecific antibodies, each of which contains nucleic acid sequences encoding a common human light chain and an Ig heavy chain with different CH3 regions and different VH specificities. Table 5 shows the transfection scheme and results.

[0185] Table 4: VH specificity inserted in different constructs

[0186]

[0187] Table 5:

[0188]

[0189] It has been previously demonstrated that the CH3 engineered Ig heavy chains encoded by constructs 1 and 2 are still able to form homodimers when expressed alone in a single cell (WO2009 / 089004). However, WO2009 / 089004 also reports that CH3 domains engineered to contain ternary charge pair mutations (such as the CH3 domains present in construct 3) are no longer able to form homodimers when expressed alone.

[0190] In the present study, these findings were only partially confirmed. In fact, the results of transfections B, C and D demonstrated the presence of full IgG in addition to a high proportion of unpaired half molecules, demonstrating partial homodimerization in the CH3 domains encoded by constructs 1 and 2. Transfections E and F also resulted in the production of full IgG in addition to unpaired half molecules, demonstrating that the ternary charge mutation of construct 3 does not completely restrict homodimerization. It was further demonstrated that the "knob" and "hole" CH3 variants of constructs 6 and 7 also form homodimers (18% homodimers are "knob-knob", 42% homodimers are "hole-hole").

[0191] It is preferred that a CH3 variant completely avoids homodimerization when it is expressed alone, so as to avoid or minimize undesirable side products (homodimers) when it is co-expressed with a second CH3 variant for heterodimerization.

[0192] Interestingly, this experiment demonstrated for the first time that it is also possible to express a mixture of bispecific antibodies in a single cell with almost no homodimers in the mixture. Transfections K and L clearly showed that the expected bispecific species BC+AB were indeed obtained (38%+47% in transfection K and 16%+60% in transfection L). In both transfections, relatively high percentages of undesirable half molecules were observed (15% half molecules A+half molecules C in transfection K and 24% half molecules A+half molecules C in transfection L). The relatively high percentage of half molecules that still exist in the transfection is attributed to the small amount of matching heavy chains in vector IV due to the imbalance of heavy chain expression in the matching pair. Therefore, in transfections S and T, the transfection was repeated with a carrier DNA adjusted in the ratio (2:1:1). This resulted in equal amounts of IgG heavy chains constituting the matching pair, as well as the absence of half IgG molecules and a pure mixture of bispecific IgG with as little as 3% homodimeric BB. Ideally, this low proportion of contaminating monospecific products should be reduced to essentially zero. Therefore, it is desirable to discover additional CH3 mutants that would result in minimal contaminating monospecific antibodies being present in a mixture of bispecific antibodies.

[0193] This study demonstrates for the first time that a substantially pure mixture of two bispecific antibodies recognizing three different target epitopes can be produced in a single cell with minimal presence of monospecific antibodies in the mixture.

[0194] Example 12: Multiple mixtures

[0195] As it has been demonstrated that it is technically feasible to produce a mixture of two bispecific antibodies recognizing three epitopes from a single cell, or a mixture of two or three monospecific antibodies from a single cell, we next explored the feasibility of controllably producing a variety of other mixtures. A fourth antibody VH region with known specificity and known to be able to pair with the human IGKV1-39 light chain was used for recloning into a vector comprising constructs 1 to 3 or 7 in Table 1, thereby producing vectors I', II', III' or X' (' refers to different specificities, compared to the corresponding vector numbering). Subsequently, the resulting vectors I' to III', X' and IV to IX were combined with other construct vectors and transfected into cells to obtain a variety of mixtures of bispecific and / or monospecific antibodies, each of which contained nucleic acid sequences encoding a common human light chain and an Ig heavy chain with different CH3 regions and different VH specificities. The various mixtures obtained from a single cell included a mixture of 2 bispecific antibodies recognizing 4 epitopes, a mixture of 2 bispecific antibodies and one monospecific antibody, or a mixture of 1 bispecific and one monospecific antibody.Table 6 shows the transfection scheme and expected results.

[0196] Table 6

[0197]

[0198]

[0199] Although, in theory, the production of all mixtures should be feasible, it is known from previous work by others that large-scale production of the classic knob-into-hole variant is limited by instability issues. Therefore, it is expected that the mixtures produced by transfecting ZA, ZB, ZL, ZM and ZN will have problems when transferred to larger scale production.

[0200] Since, the knob-into-hole variants have been reported to be unstable, and it cannot be excluded that the CH3 domain containing the "knob" or "hole" forms dimers with the charged variant or wild-type CH3 domain, the current set of constructs present in Table 1 does not allow all theoretical mixtures to be produced from a single cell on a larger scale. Therefore, it is desirable to design new CH3 variants that are engineered to preferentially form only homodimers or heterodimers, and which do not form homodimers or heterodimers with constructs 1 to 5 in Table 1 to enable co-expression in a single cell.

[0201] Example 13: Identification of new charge pair mutants

[0202] The purpose of this study is to transform the IgG CH3 region, so that when different IgG heavy chains are mixed and expressed in a single cell, only heterodimers or only homodimers are produced, wherein the newly transformed CH3 domain will not form homo or heterodimers with the known CH3 domain or wild-type CH3 domain of the transformation. Therefore, as the first step of identifying a new CH3 domain that meets the standard, many interface contact residues in the IgGCH3 domain are scanned one by one or in groups, looking for replacements that can cause the same heavy chain to repel by electrostatic interaction, that is, replacements that reduce homodimer formation. The purpose is to obtain such a series of residues, when it is replaced by a charged residue, causing the same chain to repel, so that when different IgG heavy chains are mixed and expressed, these mutations can be used to drive homo and / or heterodimer formation, thereby making the full-length IgG obtained stable and produced at a high ratio. Subsequently, by transforming the matching pair (CH3 region) of the CH3 residues in one or more IgG heavy chains, a mixture of bispecific antibodies or bispecific antibodies or monospecific antibodies is produced using the replacement identified. In addition, newly identified charge mutant pairs can be combined with existing pairs, so that multiple nucleic acid molecules encoding different heavy chains that all carry different complementary CH3 mutations can be used for expression in cells, thereby preferentially obtaining a mixture of only monospecific antibodies or only bispecific antibodies, or a mixture of limited monospecific antibodies and bispecific antibodies. The residues to be tested in this study are previously determined contact residues (Deisenhofer J., 1981; Miller S., 1990; Padlan, 1996, Gunasekaran, 2010). The basic principle of this method is to transform repulsive charges into each available pair of contact residues. Then, the samples are analyzed on non-reducing SDS-PAGE to determine the pairs that reduce dimer formation, such as the presence of a band of about 72kD. According to this method, since the repulsive electrostatic interaction between a non-matching pair may be sufficient or insufficient to produce a sufficient amount of half molecules for detection, all available pairs are screened as single mutations or in combination with single other mutations, and the mutations are combined.

[0203] According to Table 7, amino acid substitutions were introduced into the construct vector MV1057 by Geneart, and the constructs were expressed by transfection into HEK293T cells according to standard methods. IgG expression levels were measured in Octet. When two failed to produce, the mutation was considered to be detrimental to expression, and the mutation was not further studied.

[0204] Table 7: List of amino acid substitutions in various constructs prepared (EU numbering)

[0205]

[0206]

[0207]

[0208] Supernatants containing >5 μg / ml IgG were analyzed in SDS-PAGE and IgG was purified using protein A. Proteins were stained using colloidal blue. The homodimer was visible as a band of approximately 150 kD. A smaller band of approximately 75 kD indicated the presence of half molecules (see negative controls: K392D and K409D). The blot shows Figure 7 middle.

[0209] As shown in the right-hand column of Table 7, the SDS-PAGE gel results were analyzed and scored. It is believed that many residues are promising for further testing in combination, including residues Q347, S354, Y349, L351, K360, T366, T394 and V397. The selection is based on the high scores in inhibiting the formation of homodimers and the availability of contact residues that can be modified without causing problems, for example, other non-complementary charges. For example, it is known that residues F405 and Y407 have multiple interactions at the CH3-CH3 interface, including interactions with charged residues, and after introducing multiple charge mutations in these interacting residues, the interactions may have problems (see Table A). New constructs were generated in vector MV1057 (Table 8), and antibody VH regions with known specificity and known ability to pair with human IGKV1-39 light chains were used for recloning into vectors containing these new constructs (see Table 9), so that the combination can be further tested. Table 10 shows the transfection scheme and results.

[0210] Table 8:

[0211]

[0212] Table 9: VH specificity inserted in different constructs

[0213]

[0214] Table 10:

[0215]

[0216] The CH3 variant combinations were expressed and analyzed in SDS-PAGE (data not shown) and native mass spectrometry (MS). The results are summarized in Table 10. ZO transfection resulted in the highest proportion of heterodimers in the mixture (69% AC). Interestingly, in the ZO transfection, AA homodimers were absent, while a small proportion of CC homodimers (7%) was included. Mass spectrometry analysis revealed that the remaining protein in the mixture consisted of half A molecules, which may come from unequal expression of A and C heavy chains. The raw MS data from transfected sample ZO are shown in Figure 8 middle.

[0217] Surprisingly, while transfection of ZO resulted in comparable amounts of bispecific products, transfection of the reversed charge pair of ZP (L351K / T366'D vs. T366K / L351'D of ZO) did not result in similar results, with only 52% of bispecific products observed, with comparable amounts of both homodimers (30% AA and 13% CC). The explanation for this is that the negatively charged D is structurally close to T, and therefore, T366D may not be sufficiently effective to exclude itself, and therefore, T366D still forms homodimers, as actually observed.

[0218] It is conceivable that minor variations of the newly discovered T366K / L351'D pair (eg, by testing all substitutions, including the new constructs T366R and L351E) could result in similar percentages of BsAbs.

[0219] Example 14: Use of HADDOCK to design new CH3 mutants to drive efficient heterodimerization

[0220] As described in Example 13, the newly discovered charge pair T366K / L351'D increased the proportion of heterodimers in the mixture (69%), of which a small portion was an undesirable CC homodimer (7%) (L351D / L351'D), and had a considerable portion of half A molecules (24%) "contaminating" the mixture. In this example, an in silico approach was used to further deepen the understanding of the amino acid residues involved in CH3 interface interactions, thereby testing complementary substitutions in the relative CH3 regions and discovering new CH3 pairs containing complementary substitutions, which further improved effective heterodimerization while avoiding effective homodimer formation of the two heavy chains.

[0221] HADDOCK (High Ambiguity Driven protein-protein DOCKing) is an information-driven flexible docking method for modeling biomolecular complexes. HADDOCK itself is distinguished from ab initio docking methods by the fact that HADDOCK encodes information from defined or predicted protein interfaces in ambiguous interaction restraints (AIRs) to drive the docking process (de Vries et al., 2010).

[0222] The input of the HADDOCK web server consists of protein structure files, which can be crystal structures, NMR structure clusters or modeled structures. After docking or refinement, HADDOCK returns the so-called HADDOCK score, which is a weighted average of VanderWaals energy, electrostatic energy, embedded surface area energy and desolvation energy. Although it is often difficult to obtain a direct translation of experimental data, the HADDOCK score can be interpreted as representing binding energy or affinity. In addition, HADDOCK provides the structure files of the "first four" structures generated by the docking run. These structure files can be downloaded and visualized, making it possible to analyze the interactions of each residue in detail.

[0223] In this example, the interaction between the CH3 domains of the IgG1 heavy chain was investigated. The high-resolution crystal structure of the Fc portion of IgG (structure 1L6X) was used as a starting structure (http: / / www.resb.org / pdb / explore.do?structureId=116x; Idusogie, EE et al., JI 2000 (164) 4178-4184).

[0224] In Example 13, it was found that co-transfection of vectors XIII and XVI resulted in the formation of CC homodimeric contaminants (Table 10). HADDOCK was used to search for additional mutations of the T366K / L351'D pair that avoided homodimerization.

[0225] The output of HADDOCK consists of a calculated energy, a HADDOCK score (weighted average of the energy), and a group of four structure files corresponding to the four lowest energy structures found by the program. The HADDOCK score is used to compare different structures; other energies are only used to obtain an indication of what is happening in the structure (e.g., good electrostatic interactions, smaller embedding surface, higher van der Waals energy). The lower the HADDOCK score, the better. For each mutation pair, the scores of AA, AB, and BB dimers are calculated.

[0226] The mutation pair set from Example 12 was analyzed in HADDOCK to see if the calculated energy was related to the experimental data. Table 11 shows Fig. 9 All theoretical energies visible in .

[0227] Table 11:

[0228]

[0229] *This value is generally higher due to high VanderWaals energy score, probably due to steric clash of T366W / T366'W.

[0230] For the two wild-type CH3 domains, AA, AB, and BB had the same HADDOCK score, since the A and B CH3 regions were identical. In most other cases, the AB pair had the lowest score, as expected. For the T366K / L351D pair, BB scored slightly better than AB (-210.6 vs. -212.5), however, the difference was within the calculation error. The heterodimeric structures of these pairs were visible using HADDOCK. For example, construct combinations 1-2, 1-1, and 2-2 are shown in Fig.10 From these visualizations, it is clear that salt bridges are formed in the heterodimer ( Fig.10 A left-hand figure), while there is electrostatic repulsion between residues in the same chain ( Fig.10 B and C, middle and right-hand panels). Therefore, the higher HADDOCK score of the homodimer can be explained by electrostatic repulsion of the mutated interface residues. These residues are bent away from each other and do not interact with residues on the other chain, resulting in a decrease in affinity.

[0231] Table 11 and Fig. 9 This confirmed the results observed in Example 13. The T366K / L351'D AC heterodimer and the L351D / L351'D CC homodimer were formed at similar energies, explaining the presence of heterodimers and homodimers in the mixture. On the other hand, despite the presence of T366K half A molecules, the T366K / T366'K AA homodimer was barely detectable in the mixture. Table 11 and Fig. 9 It is actually shown that the HADDOCK score of the T366K / T366′K AA homodimer is higher than that of the AC heterodimer, and thus the formation of this homodimer is energetically unfavorable.

[0232] Example 15: 366 / 351 Variation

[0233] In Example 13, it is assumed that alternatives to the T366K / L351'D mutant charge pair can be designed that can have similar results in terms of the percentage of bispecific antibodies in the mixture. Alternatives can include replacing T366R, T366D, T366E, L351E, L351K, and L351R. The proportion of CC homodimers of L351D / L351'D can be reduced by generating variants of the 366 / 351 pair. In HADDOCK, all possible mutation pairs are analyzed and the resulting scores are shown in Table 12 and in Fig.11 Visualization.

[0234] Table 12

[0235]

[0236] When looking at the HADDOCK scores, it was observed that some of the mutations had a similar "pattern" when compared to T366K / L351'D. For most substitutions, the AA homodimer was found to have a higher HADDOCK score than the AB heterodimer, but the BB homodimer seemed to be as favorable as the AB heterodimer. Although the 351 residue is known to be its own "neighbor" on the other chain, that is, at the CH3-CH3 interface, residue 351 of chain A is paired with residue 351 of chain B, there is almost no negative influence of this same charge when the BB dimer is formed. Looking at the L351D / L351'D structure, the above situation is explained by the stabilizing influence of the aspartic acid bending away from each other, the naturally occurring arginine at least at position 355, and also some stabilization by the negative charge of the naturally occurring serine at position 354 (see. Fig.12 A). Mutation of these residues (S354A and R355D) provided only minor improvements. Fig.12 It is clearly seen in B that the backbone-hydrogen of A354 leads to stabilization of the homodimer. For the bispecific molecules, the T366R / L351'E pair with the lowest HADDOCK score from this series appears to be the most favorable.

[0237] Example 16: Mutations around T366K / L351'D

[0238] In the HADDOCK analysis series in this embodiment, T366K / L351'D or T366K / L351'E pairs were used as starting structures. Additional mutations on the B chain were used to calculate HADDOCK scores and energies to identify additional mutations that could further increase the predicted percentages of the bispecificity of these A and B chains. When the structure of the CH3 domain was studied using an observer (YASARA, www.yasara.org) for visualizing protein structures at the molecular level, the distances between the residues can be calculated. While doing so, it was observed that two residues, Y349 and L368, were adjacent residues that had a positive or negative impact on dimer interactions, and in this embodiment, in addition to the L351D mutation, the residues were also mutated to study the results of dimer formation on homo- and heterodimers (see Fig.13 ). Both residues appear to increase the stability of the heterodimer (lower HADDOCK score) while increasing the instability of the BB dimer (higher HADDOCK score). Glutamic acid (E) at positions 349 and 368 appears to be more favorable than aspartic acid (D). Therefore, introducing a second amino acid substitution in the B chain that already contains the amino acid substitution at position 351 appears to be more favorable for heterodimerization.

[0239] In the next set of HADDOCK analysis, the T366K / L351'D pair was used again as the starting structure. In addition to the substitutions in the B chain that further increased heterodimerization (ie, Y349D / E and L368E), additional mutations were added to the A chain that already contained the T366K substitution. Fig.14 As shown, there are several mutation pairs that appear to favor the formation of bispecific heterodimers. In the T366K-L351K / L351'D-Y349'D pair, all four mutated residues participate in heterodimer pairing, but this is not the case for T366K-L351K / L351'E-L368'E, where K351 is not directly involved in binding. However, the HADDOCK score of this latter heterodimer is -228.9, significantly lower than -214.2 for T366K / L351'E-L368'E, which can be explained by the hydrogen bonding interaction of K at position 351 (see Fig.15 ). The T366K-L351K / L351'D-Y349'D pair can be further improved by the R355'D mutation in the B chain, which results in a higher BB-HADDOCK score, but at the same time a slightly higher AB HADDOCK score. Overall, the additional L351K results in a lower AB score and similar AA and BB scores compared to the single T366K mutation in the A chain. In theory, this will result in a higher amount of bispecific heterodimers in the sample.

[0240] Depend on Fig.11 Apparently, R at position 366 drives heterodimerization more efficiently than K. Fig.13 , but now using T366R in chain A instead of T366K. It was demonstrated that combining R366 in chain A with a double mutation in chain B is disadvantageous ( Fig.16 ). This may be attributed to the larger size of this residue, interfering with other interface interactions, even though all expected salt bridges with R366 are present in the structure. Likewise, the HADDOCK score for AA homodimers is lower for R366 than for K366, which also has an adverse effect on the formation of heterodimers. Therefore, R366 in the interface was not used for further HADDOCK analysis.

[0241] According to HADDOC prediction, a total of 14 best performing pairs were selected (see Tables 13 and Fig.17 ). In some pairs, an R355D substitution was included to eliminate the stabilizing influence of the naturally occurring R355 on the L351 / L351'D interaction.

[0242] Table 13:

[0243]

[0244] Example 17: In vitro expression of bispecific

[0245] The analysis in Example 16 showed that some CH3 variants with additional mutations around the T366K / L351'D pair produced a mixture with a higher proportion of bispecific components and a potentially lower proportion of homodimeric components. These best performing pairs were selected for production and further analysis. In addition, constructs T366R and L351E were also produced. Table 14 lists the produced constructs, which were used to recloned antibody VH regions with known specificity and known to be able to pair with human IGKV1-39 light chains. The expression of IgGs containing each construct was previously reported in Example 13, and the expression was repeated with the constructs listed in Table 14. The purpose is to evaluate which of the constructs can homodimerize in the absence of a matching heterodimerization partner. Ideally, a high percentage of half bodies and a low percentage of homodimers will be formed. As a control, constructs containing previously reported charge mutations and constructs containing previously reported knob-in-hole mutations were also used to express complete IgGs by recombinant cells. The supernatant of the Protein A purification was analyzed in SDS-PAGE and the results were analyzed and scored as shown in Table 14.

[0246] Table 14:

[0247]

[0248] The results of co-expression of a common light chain with two different heavy chains carrying the amino acid substitutions of the constructs shown in Table 14 or heavy chains carrying the amino acid substitutions of the previous constructs are shown in Table 15. Expression of two different heavy chains comprising the amino acid substitutions T366K and L351'D:L368'E, respectively, resulted in about 87% bispecific AB heterodimers in the mixture, in which AA or BB homodimers were not present (combination nr. 3 of Table 15). It was observed that about 12% of the half molecules (half A) contained the T366K substitution. In addition, when the additional amino acid substitution L351K was introduced in the first heavy chain, it was found that the percentage of bispecific AB heterodimers increased. For example, co-expression of two different heavy chains comprising the amino acid substitutions T366K:L351K and L351'D:L368'E, respectively, resulted in about 92% bispecific AB heterodimers, while AA and BB homodimers were essentially absent in the mixture (combination nr. 12 of Table 15). Combinations 10 and 11 also result in a favorable distribution, with a high percentage of heterodimers and almost no homodimers. The absence of homodimers is advantageous because the portion comprising the complete IgG molecule consists only of AB heterodimers. For purification and subsequent therapeutic applications, the half molecules can be removed by standard methods (e.g., size exclusion chromatography). Therefore, in the production process for generating bispecific antibodies, the application of these newly identified charge mutants provides more advantages than known charge mutants and knob-into-hole mutants that do not exclude the presence of "contaminating" homodimeric antibodies. In addition, the T366K / L351'D:L368'E and T366K:L351ML351'D:L368'E charge pairs have an additional advantage over the previously described E356K:D399K / K392'D:K409'D and E356K:D399K / K392'D:K409'D:K439'D charge reversal pairs in that the previously described charge variants are based on the reversal of existing charges within the CH3-CH3 interface, while the newly identified charge variants are the addition of additional charge pairs (charge-charge interactions) to the CH3-CH3 interface. The introduction of additional charge pairs in the CH3-CH3 interface can further increase the stability of the interface and thereby increase the stability of the complete antibody. The same applies to the mutations used in combinations nr. 4, 5, 6, 9, 10 and 11, which also result in a favorable ratio of bispecific heterodimers in the mixture, in which a very low ratio of AA and BB homodimers is present.

[0249] Table 15:

[0250]

[0251] *Chain A carries the specificity of MF1337 (= tetanus toxoid); **Chain B carries the specificity of MF1122 (= fibrinogen)

[0252] MS

[0253] Intrinsic MS was performed on all bispecific samples. The obtained plots were analyzed in two ways to determine the relative proportions of the species present: by peak height and by peak area. Peak area is the more scientifically correct way of analysis, but since all previous analyses from other studies were based on peak height, both methods were included in the analysis for comparative purposes. The differences between the methods were within the measurement error, therefore, only the peak area values ​​were used for further measurements. Two representative spectra are shown in Fig.18 The results are summarized graphically in Figure 19 and the values ​​can be found in Table 15. In about half of the samples the total contamination of monospecific IgG was below 5%, and only in 3 cases was the value > 10%, whereas for wt IgG one would expect to find about 50% of monospecific IgG in the mixture.

[0254] Ten groups of 2 different heavy chain combinations were selected from Table 15 for further analysis. These ten combinations include combinations 1, 2, 3, 4, 5, 6, 9, 10, 11 and 12 (Table 15). These ten were selected based on the presence of a low percentage of homodimers in the mixture as determined by nMS, but also based on their overall physicochemical specificity, including yield, SDS-PAGE, and the number of mutations present in the CH3 domain.

[0255] Example 18: IgG stability analysis

[0256] In this study, a series of CH3 mutation pairs can be further analyzed for the stability of the Fc portion of the IgG molecule, which result in a high proportion of bispecific heterodimers and a very small amount (<5%) of parental IgG in the complete IgG fraction. The mutated CH3 domain used to promote heavy chain heterodimerization can have an unexpected destabilizing effect on the Fc region of IgG, which can lead to undesirable properties, such as reduced half-life in vivo, reduced effector function and / or increased immunogenicity. The newly identified charge pairs are compared with wild-type bispecifics and bispecifics containing previously identified charge mutations (chain A contains construct 1 and chain B contains construct 2). In this study, all bispecific molecules contain the same heavy and light chain variable regions to ensure that the observed effects are caused by mutations in the Fc portion of the molecule rather than by changes in the variable region.

[0257] A series of stability studies were performed on these bispecifics. These studies included spectroscopic analysis (UV-Vis absorption, fluorescence and light scattering) and microscopic analysis (optical and fluorescence microscopy with Nile red staining) to provide information on the aggregation state of the CH3 variants.

[0258] UV-Vis absorption spectra were recorded at 25°C using a dual beam, two monochromator Cary 300Bio spectrophotometer. Spectra were monitored from 250nm to 400nm using a 1cm path length. Absorption at 320nm and longer wavelengths provides information about the aggregation state of IgG.

[0259] The intrinsic fluorescence spectrum was monitored at 25°C using a FluoroMax spectrofluorometer. The fluorescence method was optimized. Fluorescence emission provides information about conformational and aggregation properties. Using a FluoroMax spectrofluorometer, the fluorescence was measured by running a synchronous scan (λ) between 400 nm and 750 nm with an integration time of 0.01 s. em =λ eχ ) The light scattering spectra were monitored at 90° at 25°C. The excitation and emission slits were optimized. For example, right angle light scattering can distinguish between IgG samples without and with 5% dimer.

[0260] For fluorescence microscopy with Nile Red staining, Nile Red ethanol solution was added to the sample just before the assay. The sample was filled onto a microscope slide and analyzed by fluorescence microscopy. The particles were counted. The lower limit of the size of particles observable by fluorescence microscopy was about 0.5 μm.

[0261] Applying stress on proteins, such as temperature, pH and mechanical stress or denaturants can lead to conformational changes (e.g., unfolding) and / or aggregation. As previously reported, charge engineered bispecific antibodies reduce the melting temperature of the modified CH3 (Gunasekaran 2010), these studies aimed to distinguish the novel charge mutants of the present invention from existing known charge mutants.

[0262] Octet was used to explore thermal stability studies using protein A biosensors and by utilizing FcRn bound to IgG. Using a PCR machine, samples were incubated for 1 hour at 4, 50, 55, 60, 65, 70, and 75°C at a concentration of 100ug / ml (in PBS) to detect the thermal stability of CH3-engineered IgG. Next, the sample was slowly cooled to 25°C over a 15-minute period and maintained at that temperature for 2 hours, and then stored at 4°C overnight. Precipitated antibodies were removed by centrifugation, and thereafter, the total IgG concentration of soluble antibodies was determined by Octet using protein A biosensors (diluted to 1 / 10 in PBS). Using Octet, experiments measuring the binding of CH3-engineered IgG to FcRn were explored. Protein L biosensors were used to bind the light chain of IgG to the sensor, followed by incubation with FcRn in solution, or anti-pentahistidine biosensors were used to bind histidine-tagged FcRn proteins, followed by incubation with the target IgG. These methods can be more sensitive than using protein A biosensors and can also be used for thermal stability studies. In addition, the serum stability of all samples was analyzed. Briefly, the (modified) IgG samples were incubated in human serum at 37°C and the control samples were kept at 4°C. After 1, 2, 3 and 4 weeks, the samples were centrifuged to remove the precipitated IgG. Subsequently, the samples were titrated in antigen-specific ELISA to determine the relative amount of functional IgG. Purified control antibodies freshly spiked in human serum were used as reference.

[0263] Example 19: Stability Analysis

[0264] In previous experiments, a high percentage of bispecific antibodies was obtained by co-expression of two different heavy chains containing CH3 mutations and a common light chain (Example 17).

[0265] From table 15, select eight groups of 2 different heavy chain combinations for further analysis.These eight combinations include combination 3,4,5,6,9,10,11 and 12 (table 15).In this study, analyze these eight combinations, focus on the stability of the Fc part of IgG.As a control, include wild-type bispecific (that is, no CH3 mutation) and / or bispecific based on previously reported CH3 charge mutation.Note that for wild-type bispecific, when not using the means for preferentially tending to heterodimer, make 2 kinds of heavy chains and common light chain coexpression.Therefore, these "wild-type bispecifics" represent the mixture of AA, AB and BB.In this study, all bispecifics are designed to carry the same VH / VL combination, ensuring that the observed impact is caused by the mutation in the Fc part of the molecule rather than the change in the Fab part.

[0266] It is hypothesized that the mutation pairs used to promote heterodimeric pairing of two different heavy chains may be associated with unexpected structural or other destabilizing effects on the Fc region of IgG. Due to the presence of these mutations, this will subsequently lead to undesirable problems, further limiting clinical development, such as reduced in vivo half-life, reduced effector function and / or increased immunogenicity.

[0267] Thermal stability

[0268] Applying stress, for example, increasing or decreasing temperature, can lead to conformational changes (e.g., unfolding) and / or protein aggregation. The bispecific molecules from combinations 3 to 6 and 9 to 12 (Table 15) as well as wild-type bispecific molecules and bispecific molecules obtained when constructs 1 and 2 (E356K: D399K / K392D ': K409D 'combination, also referred to as "charge reversal" pairs) were incubated at a concentration of 100 μg / ml (in PBS) using a PCR machine at 4, 60, 62.5, 65, 67.5, 70 and 72.5 ° C for 1 hour to determine the thermal stability of CH3 engineered IgG. Next, the samples were slowly cooled to 25 ° C over a period of 15 minutes and kept at this temperature for 2 hours, and then stored at 4 ° C overnight. The next day, the precipitated antibodies were removed by centrifugation (18,000 rpm, 4°C, 20 minutes), and then the total IgG concentration of soluble antibodies (diluted 1 / 10 in PBS) was determined by Octet using a protein A biosensor. The results are shown in Fig. 20 Compared to the wild-type bispecific antibody (squares), the control CH3-engineered bispecific antibody (charge-reversed E356K:D399K / K392D':K409D' combination (triangles)) was observed to have reduced thermal stability. Compared to the wild-type bispecific antibody, the bispecific molecules from combinations 3 to 6 and 9 to 12 (diamonds) also showed reduced thermal stability. However, three combinations clearly showed improved stability compared to the control CH3-engineered bispecific antibody. The bispecifics of combinations 9, 10 and 11 were significantly more stable than the other CH3-engineered (charge-reversed) bispecifics and were as stable as the wild-type bispecific measured at the highest temperature.

[0269] Freeze-thaw stability

[0270] In order to determine the stability of CH3-modified IgG molecules upon repeated freezing and thawing, bispecific molecules from combinations 3 to 6 and 9 to 12 (Table 15) as well as wild-type bispecific molecules and bispecific molecules obtained when using constructs 1 and 2 (E356K:D399K / K392D':K409D' combination, also referred to as charge reversal pairs) were exposed to ten subsequent freeze-thaw cycles, which were performed as follows: the sample was placed at -80°C for at least 15 minutes until it was completely frozen. Subsequently, the sample was thawed at room temperature. When the sample was completely thawed, the freeze-thaw cycle was repeated. After 10 freeze-thaw cycles, the precipitated antibodies were removed by centrifugation (18,000 rpm, 4°C, 20 minutes), after which the total IgG concentration of soluble antibodies (diluted 1 / 10 in PBS) was determined by Octet using a protein A biosensor. The freeze-thaw stability test was repeated three times. The results are shown in Fig.21 It was observed that the control charge-reversed CH3 engineered bispecific antibody showed a slight decrease in stability compared to the wild-type bispecific. In contrast, the bispecific molecules from combinations 3, 4 and 9 showed a slight improvement in stability compared to the wild-type bispecific molecule. Overall, it can be concluded that the stringent conditions of freeze-thaw cycling do not cause major stability issues for the CH3 engineered variants.

[0271] In vitro serum stability

[0272] Bispecific molecules from combinations 3 to 6 and 9 to 12 (Table 15), as well as wild-type bispecific molecules and charge-reversed bispecific molecules, were incubated in 10% human serum at 37°C to determine the stability of CH3 engineered IgG in serum maintained at 37°C. Control samples were maintained in human serum at 4°C. After 1, 2 or 5 days, the precipitated antibodies were removed by centrifugation. Subsequently, the samples were titrated in a fibrinogen-specific ELISA to determine the relative amount of functional IgG. Purified control antibodies freshly spiked in human serum were used as reference.

[0273] The fibrinogen ELISA data indicated that all samples were fairly stable in 10% human serum at 37°C for 5 days. At lower IgG concentrations, the stability of the bispecific molecules from combinations 4 and 5 appeared slightly less stable, especially at T=1 and T-2, but at the end of this experiment, there were only minimal differences (see Fig. 22 ).

[0274] Example 20: Other stability tests

[0275] Another series of analytical methods were used to evaluate the stability of variant IgGs. Bispecific molecules from combinations 3 to 6 and 9 to 12 (Table 15) as well as wild-type bispecifics (AA, AB and BB), each parent antibody (AA and BB) and bispecific molecules obtained when constructs 1 and 2 (E356K: D399K / K392D': K409D' combination (charge reversal pair)) were used as samples in these stability assays. All IgGs were diluted to 0.2 mg / ml and subjected to several stress conditions (2 days at 50°C, 2 weeks at 40°C, 5× freeze-thaw) in order to be able to distinguish between different samples. It was noted that these high stress levels led to a situation in which one of the parent antibodies used in all bispecifics (BB parent, carrying two 1122Fabs) became unstable. Aggregation of the protein was detected by UV absorption at 50°C for 2 days. This indicates that the stress conditions cannot distinguish between the instability of Fab and CH3 in the bispecific, and the data obtained by incubation at 50°C should be used with caution.

[0276] The results are summarized in Table 16. The analytical methods used included:

[0277] - Fluorescence microscopy using Nile Red ("Nile Red Particles" in Table 16); to observe the amount of particles > 0.5 μm after addition of Nile Red dye.

[0278] - UV spectrometry at 350 nm ("UV 350 nm"); changes in the absorption at wavelengths > 320 nm give information about the aggregation state of the protein.

[0279] - 90° light scattering at 400 nm ("LS 400 nm"); a sensitive technique for observing changes in protein aggregation (e.g., the difference between monomers and IgG dimers).

[0280] - Intrinsic fluorescence; upon a change in the environment (e.g., unfolding), the fluorescence wavelength maximum and intensity of aromatic residues in a protein change.

[0281] -1,8-ANS fluorescence spectroscopy; 1,8-ANS binds to cationic groups through electrostatic interactions, forming ion pairs, and changes in protein structure and / or conformation can be detected.

[0282] UV-VIS spectroscopy

[0283] UV-Vis absorption spectra were measured at 25°C in different quartz cuvettes (e.g., black low volume Hellma cuvettes with a path length of 1.0 cm and clear Hellma cuvettes of 0.2 cm x 1.0 cm) using a dual beam, two monochromator Cary 300Bio spectrophotometer from Varian. With a path length of 1.0 cm, the spectrum between 220 nm and 450 nm was monitored. The absorption around 280 nm provides information about the protein concentration. The region between 320 nm and 450 nm can provide information about the aggregation state of the sample.

[0284] 90° light scattering

[0285] The 90° light scattering spectroscopy method was developed to study protein aggregation and was performed as described in Capelle, 2005; Demeule, 2007a. The fluorescence intensity was measured using a FluoroMax spectrofluorometer (Spex, Instruments SA, Inc. UK) by running a synchronized scan (λ) between 400 nm and 750 nm with an integration time of 0.01 s. em =λ eχ ), monitoring the 90° light scattering spectrum at 25°C. Different slit settings were tested to find the optimal conditions. After optimization, the same slit setting was used for all measurements.

[0286] Steady-state fluorescence emission

[0287] The fluorescence emission of tryptophan, tyrosine and phenylalanine residues gives information about the local environment of these fluorophores. Changes or differences in hydrophobicity and / or rigidity are determined. In general, a more hydrophobic and stringent environment leads to an increase in fluorescence intensity and a blue shift in the emission maximum. Intrinsic fluorescence spectroscopy can provide information about the current state of the protein and monitor changes in physical and chemical properties. More information about tyrosine and tryptophan fluorescence can be found in the book of Lakowicz [Lakowicz, 2006].

[0288] Fluorescence emission and excitation spectra were recorded in different quartz cuvettes at 25°C. The samples were excited at different wavelengths. Integration time and slit settings were optimized. After optimization, the same integration time and slit settings were applied to all samples.

[0289] Fluorescence microscopy with Nile red staining

[0290] A Nile red staining method was developed to visualize protein aggregation and was performed as described in Demeule et al., 2007b.

[0291] Microscopy observations were performed on a Leica DM RXE microscope (Leica Microsystems GmbH, Wetzlar, Germany) equipped with a mercury lamp. Images were acquired with a Sony NEX-5 camera and its firmware. Objectives were 10×, 20×, and 40×. For microscopy examinations, a fixed distance of 0.1 mm between the slide and the cover glass was used. The size of the 4x4 grid was 1 mm x 1 mm, equivalent to 0.1 μL.

[0292] 1,8-ANS fluorescence spectroscopy

[0293] 1-Anilinonaphthalene-8-sulfonic acid (1,8-ANS) is an uncharged, small, hydrophobic fluorescent probe (Mw 299.34 Da) used to study membrane surfaces and proteins.

[0294] 1,8-ANS is essentially non-fluorescent in water and becomes significantly fluorescent only when bound to membranes (quantum yield -0.25) or proteins (quantum yield -0.7). This property of 1,8-ANS makes it a sensitive indicator of protein folding, conformational changes, and other processes that modify probes upon exposure to water. References to 1,8-ANS can be found in Molecular Probes, www.probes.com Found on the Internet homepage.

[0295] Fluorescence emission spectra of 1,8-ANS were recorded using a FluoroMax spectrometer. A direct comparison of 1,8-ANS fluorescence between IgGs was not performed. Each IgG may have a different number of 1,8-ANS binding sites, so comparisons cannot be made. In principle, the lower the fluorescence, the fewer 1,8-ANS molecules are bound to the antibody. Changes in 1,8-ANS fluorescence intensity and emission wavelength caused by stress were evaluated.

[0296] Table 16: Summary of the results of different forced degradations on each IgG sample after dilution to 0.2 mg / ml. The color of the cell indicates the change between T=0 and after stress: dark grey=large change, light grey=small change, colorless=no change (=stable).

[0297] * "Combination #" refers to the combination of mutations listed in Table 15; **Very small particles by fluorescence microscopy, the relevance of these particles is unknown; 2d4°C = 2 days at 4°C; 2d50°C = 2 days at 50°C; 2w4°C = 2 weeks at 4°C; 2w40°C = 2 weeks at 40°C; T0 = start of experiment; 5FT = 5 freeze-thaw cycles

[0298]

[0299]

[0300] In summary, these data show that each IgG sample is remarkably stable. Several harsh stress conditions (e.g., 2 days at 50°C) are required to produce measurable differences between the tested samples. Under these conditions, samples of combination #9 and #10 appear to aggregate more than the other samples.

[0301] For stability between proteins, the biggest differentiating factors were freeze-thaw cycles and temperature rise. Taking into account the very stringent stress factor of incubation at 50°C, the T366K / L351E, Y349E (combination #4) and T366K, L351K / L351D, Y349E (combination #11) variants were the two most stable proteins in the group, followed by T366K, L351K / L351D, Y349D (combination #10) and T366K, L351K / L351D, L368E (combination #12).

[0302] Example 21: Native MS of Ratio Experiment; Transfection Ratios 1:5 to 5:1

[0303] More detailed ratiometric experiments were performed to learn more about the behavior of CH3 mutated IgGs, in particular the T366K:L351K / L351D':L368E' combination (from now on referred to as KK / DE or DEKK) in skewed transfection mixtures.

[0304] The previously used antibody VH regions known to be able to pair with the common human IGKV1-39 light chain were used for recloning into constructs 1, 2, 68 and 69, thereby generating vectors I to V of Table 17. Subsequently, vectors I to V, each of which contains nucleic acid sequences encoding a common human light chain and an Ig heavy chain with a different CH3 region and a different antigen specificity, were transfected into cells at different transfection ratios as shown in Table 18. The results are shown in Fig.23 middle.

[0305] Table 17:

[0306]

[0307] Table 18:

[0308]

[0309] *Due to a technical error, this sample was not measured.

[0310] Fig.23A and B show that in all cases, for the DEKK mutation combination, when there is an excess of A or C (A or C is on the "DE side" and B is on the "KK side"), AB or BC is formed, but the remaining A or C exists as a mixture of homodimers and half bodies. However, when there is an excess of B (B is on the "KK side" and A or C is on the "DE side"), there is a clear difference. AB or BC is still formed, but the remaining B is basically not present as a homodimer, only half bodies are formed. The percentage is measured again by the peak height Nota bene: peaks detected in the range of 2% or less are below the threshold that can be accurately measured using nMS technology. Therefore, measurements of <2% are considered to be within the noise level of the analysis and are therefore ignored. Surprisingly, excess B only leads to a higher percentage of half body B. In particular, when the ratio of A:B is 1:3 and 1:5, a higher percentage of half body B ( Fig.23 A and B), indicating that the CH3 mutations on the KK side are not conducive to homodimerization. The absence of homodimers provides a key advantage because this "KK side" of the DEKK combination can be selected to introduce specificity that may have known adverse effects when present as a homodimer (e.g., cMET or CD3 antibodies are known to have undesirable adverse side effects when present as bivalent homodimers in therapeutic compositions).

[0311] For different DE:KK ratios, the observed findings were opposite to those of the control charge-reversal CH3 mutations in vectors IV and V. Fig.23 C shows that in all cases, for the E356K:D399K / K392D':K409D' mutation combination, when there is excess A (A is on the "K392D:K409D side"), the remaining A exists as a mixture of homodimers and half-bodies, but similarly, in all cases, when there is excess B (B is on the "E356K:D399K side"), the remaining B exists as a mixture of homodimers and half-bodies. Even at higher ratios of 1:3 and 1:5, half-body B is not observed, but homodimers are present, indicating that the E356K:D399K side is not as unfavorable for homodimerization as the KK side of the DEKK combination.

[0312] In summary, the DEKK mutation combination provides a clear benefit over the charge-reversal CH3 mutation in that one of the chains of the heterodimer does not form a homodimer.

[0313] Example 22: Multiple mixtures using DEKK combinations

[0314] Since it has been demonstrated that DEKK mutation combinations drive the formation of highly pure bispecific IgG molecules ("AB"), we then explored the feasibility of controllably producing more complex antibody mixtures (e.g., "AB and AA" or "AB and AC" mixtures) from one cell. The previously used model Fabs were introduced into vectors containing "DE constructs" or "KK constructs", and various combinations of these vectors were co-expressed to produce mixtures, thereby demonstrating the versatility of this technology. The model Fabs MF1337 (tetanus toxoid), MF1122 (fibrinogen), and MF1025 (thyroglobulin) were selected based on their overall stable performance, good expression levels, and the quality difference between IgGs containing these Fabs (see Table 19).

[0315] Table 19:

[0316] Specificity Fab Name IgG quality Δ Quality MF1122 Tetanus (A) (MF)*1337 146747.03 +1842.05 Fibrinogen (B) (MF)1122 144904.98 0 Thyroglobulin (C) (MF)1025 144259.87 -645.11

[0317] *MF = Merus Fab, names, e.g., MF1337 and 1337, are used interchangeably.

[0318] Table 20: Transfection protocol:

[0319]

[0320] SDS-PAGE analysis proved that most samples were mainly composed of whole IgG, with a small percentage of half bodies in some cases. In addition, on non-reducing gels, many samples showed two bands at ca.150 kDa, reflecting the presence of two different IgG species in the samples. Also on reducing gels, two heavy chain bands were visible in some samples (data not shown).

[0321] Native MS was performed on all samples and the percentage of observed species was calculated based on peak height (% observed species in Table 20). The results are shown in Fig.24 In all eight samples of three heavy chain co-expression, two main peaks corresponding to the expected species were observed. In two of these samples (transfection 2 and 4) and transfection 11, a small amount of contaminating DE-DE homodimer was observed. In most samples, very little half-body (less than 2%) was detected, which, as mentioned above, will not be a problem because they can be easily separated from the full-length IgG part. After nMS, it was found that the IgG mass observed in sample 11 corresponds to different species, rather than expected, and it is inferred that this is attributed to transfection error, that is, in sample 11, it is obvious that 1025-DE is co-transfected with 1337-KK, rather than 1122-KK.

[0322] IgG samples were further tested in sandwich ELISA to confirm the functional presence of the desired specificity. ELISA plates were coated with fibrinogen or thyroglobulin and detected using fluorescein-labeled thyroglobulin or tetanus toxoid. Detection antigens were labeled with fluorescein (Pierce NHS-fluorescein antibody labeling kit, catalog number #53029) according to the manufacturer's instructions. Subsequently, fluorescein-labeled antigens were detected by FITC-conjugated anti-fluorescein antibodies (Roche diagnostics, catalog number #11426346910).

[0323] The results (OD450 values) of the bispecific ELISA are summarized in Table 21. The gray cells indicate the expected species for each transfection. In summary, the results were consistent with the expected results, except for those indicated in italics or bold. In transfections 1 to 3, the supposed "negative" wells for species BC (tr. #1 and 2) or AC (tr. #3) demonstrated significant background signals. It is known from previous studies that bispecific ELISAs can suffer from higher background levels. These background levels can also be caused by potential presence of half bodies in the sample. Clearly, the bispecific ELISA results did confirm that an error occurred in transfection 11, as species AC (bold values) were detected instead of BC.

[0324] Table 21: OD450 values ​​from dual specific ELISA

[0325]

[0326] Example 23: Improved cocktail of two bispecific antibodies (AB and CD) recognizing four different epitopes from a single cell

[0327] In Example 12, it was assumed that the mixtures expected to be generated by transfection of ZA or ZB would become problematic when transferred to large-scale production, since the knob-into-hole variants were reported to be unstable, and the formation of dimers between the CH3 domains comprising the "knob" or "hole" and the charge-engineered CH3 domains could not be excluded. As demonstrated in the above examples, the new charge pair mutations were found to preferentially drive heterodimerization, while homodimers were hardly formed, and therefore, CH3 domain-containing polypeptide chains comprising these new charge pair mutants could be expressed in cells together with previously known charge-engineered CH3 domain-containing polypeptide chains or potentially with SEED bodies, and could result in preferential formation of only two bispecific molecules.

[0328] As can be seen from the above examples, the combination of DEKK mutations is excellent for producing one bispecific (AB) or two bispecifics (AB plus AC) by cloning cells in which the dimerization of the heavy chain is driven by the CH3 domain. However, using only one vector set of complementary CH3 mutations limits the number of possible types of mixtures that can be produced. If a second "orthogonal" vector set is used in combination with DEKK, more complex mixtures of IgG and / or bispecifics can be produced, for example, "AB and CD" or "AB and CC" mixtures. When combining two vector sets, an important condition is that the heavy chains expressed by the two different CH3-modified vector sets cannot form "cross" dimers, that is, the heavy chains produced by one vector set dimerize with the heavy chains expressed by the other vector set into full IgG.

[0329] To test the potential formation of "crossover" dimers, in silico analysis using HADDOCK was performed to further understand whether possible pairing would occur between the wild-type CH3 domain and the CH3 domain containing the DE- or KK mutations. Similarly, potential pairing between the wild-type CH3 domain and the CH3 domain containing the E356K, D399K or K392D, K409D mutations, as well as potential pairing between the wild-type CH3 domain and the CH3 domain containing the knob-into-hole mutations and any combination of the above, was analyzed. The combinations of CH3 mutants analyzed in HADDOCK are listed in Table 22, and the obtained HADDOCK scores are summarized in Fig.25 middle.

[0330] Table 22: CH3 variants analyzed in HADDOCK, with a single letter code assigned to each heavy chain carrying a CH3 variant. * For consistency, the wild-type chains are named "C" and "D"; ** Charge-reversal variants are named "A" and "B" when combined with the knob-into-hole variants, and "C" and "D" when combined with the DE / KK variants.

[0331]

[0332] Fig.25 It was shown that, based on these HADDOCK predictions, combining the CH3 combinations of DEKK with the charge-reversed CH3 combinations is most likely to successfully form the desired combinations of two bispecifics (AB and CD) without contaminating byproducts (especially AC, AD, BC, BD) when co-transfected in a single cell. Fig.25It can be seen that these undesirable bispecific species AC, AD, BC and BD have relatively high HADDOCK scores, while the desired AB and CD species have the lowest HADDOCK scores. Of course, when a DEKK or charge-reversed CH3 combination is introduced into a construct carrying the same specificity (e.g., "C" on the DE side, "C" on the KK side, "A" on the E356K, D399K side and "B" on the E356K, D399K side, or "A" on the DE side, "B" on the KK side, "C" on the E356K, D399K side and "C" on the E356K, D399K side), this will result in the production of mainly CC and AB when co-expressed in cells.

[0333] In contrast, when looking at the predictions for co-expressing DEKK with wild-type, it can be seen that the HADDOCK scores for AC and AD are both lower than that for CD, suggesting that AC and AD are very likely contaminants when attempting to generate a mixture of AB and CD by co-expressing vectors encoding a combination of CH3s of DEKK with vectors encoding wild-type CH3. Finally, co-expression of DEKK or a charge-reversal variant with a knob-into-hole variant is predicted to result in undesirable bispecific variants with relatively low HADDOCK scores, i.e., there is a higher likelihood of generating these undesirable species when co-expressed.

[0334] It follows that combining the CH3 combination of DEKK with the charge-reversed CH3 combination (E356K, D399K / K392'D, K409D') is ideally suited to obtain substantially pure "AB and CD" and / or "AB and CC" antibody mixtures.

[0335] Next, a mixture of two bispecific antibodies recognizing four targets / epitopes (AB and CD) and a mixture of a bispecific antibody and a monospecific antibody recognizing three targets / epitopes (AB and CC) was produced by putting the above into practice. These mixtures are produced using four different VHs, all of which can be paired with the common light chain IGVK1-39, but each VH / VL combination has a different specificity. (Expected) species should have a sufficient mass difference (i.e. >190Da) to enable intrinsic MS analysis. Four separate VHs were selected, and these VHs should have such a mass that the expected species can be identified and separated by nMS during co-transfection. In addition, the mass difference of the selected four VHs is also large enough to identify most of the possible contaminants in the mixture except for the two desired species. The selected VHs are listed in Table 23.

[0336] Table 23:

[0337]

[0338] The 4 different VHs were cloned into vectors containing either a "DE" or "KK" construct or a charge reversal construct, and several co-transfections were performed as shown in Table 24. NB: As usual, all vectors also contained nucleic acid encoding a common light chain IGKV1-39. As previously described, when combining two vector groups, an important condition is that the heavy chains expressed by the two different CH3-modified vector groups cannot form "cross" dimers, i.e., the heavy chains produced by one vector group dimerize with the heavy chains expressed by the other vector group into full IgG. Control transfections were performed to test this potential formation of "cross" dimers between heavy chains containing charge reversal mutations and heavy chains containing DE or KK mutations.

[0339] Table 24:

[0340]

[0341] Table 25 further provides an overview of the quality of expected species and possible contaminants in transfections #9 to 11 in Table 24.

[0342] Table 25: For each of transfections #9 to 11, the species were sorted by mass, using the mass above to calculate the mass difference. Grey cells: expected (and desired) species; italics: mass differences too small to be separated in nMS analysis. *Species: single letter indicates hemibody; two letter code is intact IgG.

[0343]

[0344] All purified protein samples obtained from transfections #1 to #11, including three control samples ( Fig.26 In addition, nMS analysis was performed on protein samples from transfections #9 to #11 to identify all species in the samples. Fig.26 As can be seen, transfections #3 and #4 resulted in the expected mismatch between the "KK" construct and either "E356K:D399K" or "K392D:K409D", and the amount of half-bodies exceeded the amount of full IgG molecules in the protein samples from these transfections. Transfections #7 and #8 resulted in protein samples in which half-bodies and full IgG were present in approximately equal amounts. However, it could not be inferred from SDS-PAGE whether the full IgG represented a DE / DE dimer, a DE / E356K:D399K dimer or a DE / K392D:K409D dimer. Significantly, almost no half-bodies were observed in the protein samples from transfections #9 to #11.

[0345] nMS analysis of transfection #9 and #11 showed Figure 27A-27CThe percentages of expected and contaminating species were calculated by peak height. It was demonstrated that for transfection #9, the expected species "AB and CD" accounted for 97% of the mixture (30% AB and 67% CD), while there was only about 3% contaminating BD ( Fig.27A , Fig.27B ). For transfection #11, the expected species "AB and CC" accounted for 94% of the mixture (33% AB and 61% CC), while only as little as about 6% of contaminating BC (4.1%) and AC (1.8%) were present ( Fig.27C ). These data suggest that it is indeed possible to generate more complex mixtures of IgG and / or bispecifics, such as "AB and CD" or "AB and CC" mixtures, when a second "orthogonal" vector set is used in combination with DEKK. Combining the charge-reversed construct with the DEKK construct results in only very limited formation of "crossover" dimers. By adjusting the transfection ratio, it is expected that the low percentage of these contaminating byproducts can be reduced even further.

[0346] Example 24: Single-dose pharmacokinetic study in mice

[0347] In this study, the pharmacokinetic parameters of three different IgG batches were determined and compared to study the pharmacokinetic (pK) behavior of bispecific antibodies carrying a combination of DEKK mutations in their CH3 regions. The three IgG batches included 1) wild-type anti-tetanus toxoid parent antibody 1337:1337 (two MF1337 Fabs on a wild-type Fc backbone); 2) wild-type anti-tetanus toxoid parent antibody 1516:1516 (two MF1516 Fabs on a wild-type Fc backbone); 3) CH3 engineered bispecific anti-tetanus toxoid antibody 1516:1337, which carries a combination of DEKK mutations in its Fc region (MF1516 Fab on the DE side, MF1337 Fab on the KK side).

[0348] The parent antibodies 1337:1337 and 1516:1516 included in the DEKK bispecific product were selected based on specificity because there was no pre-dose serum response against these antibodies in several mouse strains based on previous studies. NB: The presence of a pre-dose serum response certainly invalidated this study. In addition, there was sufficient mass difference between the parent antibodies to enable identification of the 1337:1337 (wt Fc), 1516:1337 (DEKK Fc) and 1516:1516 (wt Fc) species by nMS. Three IgG batches were prepared as described above, but the DNA for transfection was prepared using an endotoxin-free maxiprep kit to ensure that the amount of endotoxin was as low as possible. Subsequently, these batches were tested for protein concentration, aggregation level, endotoxin level and bispecific product percentage. It was demonstrated that the acceptance criteria for subsequent use of the IgG batch were met in the pK study, i.e., IgG concentration after gel filtration > 0.3 mg / mL, aggregation level < 5%, endotoxin level < 3 EU / mg protein and that the DEKK batch contained > 90% bispecific IgG.

[0349] The native mass spectra of the gel filtered samples showed that the expected species were present in high percentages. In sample 1516:1337, a small amount of DE:DE homodimer was detected, which was estimated to be ca. 2% ( Fig.28 ). It can be concluded that the three IgG batches are suitable for use in the pK study.

[0350] 1 mg / kg human IgG (5 ml / kg immunoglobulin solution / kg body weight) was administered to 3 groups of female C57BL / 6J mice (Harlan, The Netherlands) to compare the pK parameters between the three batches. At the time of administration, the animals were 7 to 8 weeks old and weighed approximately 18 to 20 grams. Blood samples were collected before administration and 15 minutes, 60 minutes, 2 hours, 4 hours, 8 hours, 24 hours, 48 ​​hours, 96 hours, 168 hours, 268 hours and 336 hours after administration. Serum samples were prepared and stored at <-20°C until analysis. Each group consisted of 3 subgroups of 4 mice, i.e., 12 mice / group. Six time points were sampled from each mouse. The welfare of the animals was maintained according to the basic principles governing the use of animals in experiments in the European Community (Directive 86 / 609 / EEC) and Dutch legislation (Animal Experimentation Act, 1997). This study was also conducted in accordance with the standards for humane care and use of laboratory animals issued by the Office of Laboratory Animal Welfare of the National Institutes of Health, identification number 45859-01 (expiration date: April 30, 2015).

[0351] Group 1 mice received full-length monospecific IgG 1516:1516 antibody (triangles); Group 2 mice received full-length monospecific IgG 1337:1337 antibody (squares); Group 3 mice received full-length bispecific IgG 1516:1337 antibody with DEKK engineered CH3 region (1516 on the DE side, 1337 on the KK side) (diamonds);

[0352] The ELISA assay was used to quantify the monoclonal human antibodies in mouse serum using a quantitative human IgG ELISA (ZeptoMetrix, NY USA; ELISA kit nr. 0801182). Briefly, the ELISA assay is based on the following principle: human monoclonal antibodies bind to anti-human IgG coated in a 96-well ELISA plate. Subsequently, the bound antibodies are visualized using a polyclonal anti-human IgG antibody conjugated to horseradish peroxidase (HRP). The optical density (OD) of each well is proportional to the amount of antibody in the serum sample. The results are shown in Fig.29 In the present study, it was observed that the serum levels of both the bispecific full-length IgG antibody carrying the combination of DEKK mutations and its parental monospecific antibody were significantly similar. It was concluded that the CH3 mutations present in the DEKK bispecific antibody did not change either the stability or the half-life, and the DEKK variant behaved like a wild-type IgG.

[0353] References

[0354] Deisenhofer J., Biochemistry 1981(20)2361-2370;

[0355] Miller S., J.Mol.Biol.1990(216)965-973;

[0356] Padlan, Advances in Protein Chemistry 1996(49)57-133

[0357] Ellerson JR., et al., J. Immunol1976(116)510-517;

[0358] Lee and Richards J.Mol.Biol.1971(55)379.

[0359] Gunasekaran et al J.Biol.Chem.2010(285)19637-19646

[0360] De Vries Nature Protocols 2010(5)883

[0361] Kabat et al,(1991)

[0362] Mammalian Cell Biotechnology:a Practical Approach(M.Butler,ed.,IRLPress,1991

[0363] Merchant Nature biotechnology 1998(16)677

[0364] Ridgeway Protein Engineering 1996(9)617-621.

[0365] Davis JH.Et al.,Protein Engineering,Design&Selecttion 2010(23)195-202

[0366] Papadea and check.Crit Rev Clin Lab Sci.1989;27(1):27-58.

[0367] Tissue Culture,Academic Press,Kruse and Paterson,editors(1973)

[0368] Ionescu et al.,J.Pharm.Sci.2008(97)1414)

[0369] Current protocols in Protein Science 1995,coligan JE et al.,WingfieldPT,ISBN 0-471-11184-8,Bendig 1988.

[0370] Capelle,M.A.H.,Brugger,P.,Arvinte,T.Vaccine 23(2005),1686-1694.

[0371] Demeule,B.,Lawrence,M.J.,Drake,A.F.,Gurny,R.,Arvinte,T.Biochim.Biophys.

[0372] Acta 1774(2007a),146-153.

[0373] Demeule, B., Gurny, R., Arvinte, T., Int. J. Pharm 329 (2007b), 37-45.

[0374] Lakowicz.JR, Principles of fluorescence spectroscopy; Second Edition Kluwer Academic / Plenum Publishers, New York, Boston, Dordrecht, London, Moscow, (2006) ISBN 0-306-46093-9.

[0375] The following content corresponds to the original claims in the parent application and is now incorporated herein as part of the specification:

[0376] 1. A method for producing at least two different immunoglobulin-like molecules from a single host cell, wherein each of the two immunoglobulin-like molecules comprises two CH3 domains capable of forming an interface, the method comprising providing in the cell:

[0377] a. a first nucleic acid molecule encoding a first polypeptide chain containing a CH3 domain,

[0378] b. a second nucleic acid molecule encoding a second polypeptide chain containing a CH3 domain,

[0379] c. a third nucleic acid molecule encoding a third polypeptide chain containing a CH3 domain, and

[0380] d. a fourth nucleic acid molecule encoding a fourth CH3 domain-containing polypeptide chain, wherein at least two of the nucleic acid molecules have means for preferentially pairing the first CH3 domain-containing polypeptide with the second CH3 domain-containing polypeptide and the third CH3 domain-containing polypeptide with the fourth CH3 domain-containing polypeptide chain, the method further comprising the steps of culturing the host cell and allowing the at least four nucleic acid molecules to express and harvesting the at least two different immunoglobulin-like molecules from the culture.

[0381] 2. The method of claim 1, further comprising providing the host cell with a nucleic acid molecule encoding a common light chain.

[0382] 3. The method of claim 1 or 2, wherein the first CH3 domain-containing polypeptide chain comprises an amino acid substitution of T366K, and the second CH3 domain-containing polypeptide chain comprises an amino acid substitution of L351D.

[0383] 4. The method of claim 3, wherein the first CH3 domain-containing polypeptide chain further comprises an amino acid substitution of L351K.

[0384] 5. The method of claim 3 or 4, wherein the second CH3 domain-containing polypeptide chain further comprises an amino acid substitution selected from the group consisting of Y349E, Y349D and L368E.

[0385] 6. The method of claim 5, wherein the second CH3 domain-containing polypeptide chain further comprises an amino acid substitution L368E.

[0386] 7. The method of any one of items 1 to 6, wherein the third CH3 domain-containing polypeptide chain comprises amino acid substitutions E356K and D399K, and the fourth CH3 domain-containing polypeptide chain comprises amino acid substitutions K392D and K409D.

[0387] 8. The method of any one of items 1 to 7, wherein each of the CH3 domain-containing polypeptide chains further comprises a variable region that recognizes a target epitope.

[0388] 9. The method according to item 8, wherein the four variable regions of the four CH3 domain-containing polypeptide chains each recognize a different target epitope.

[0389] 10. The method of claim 8, wherein the variable region of the first CH3 domain-containing polypeptide chain and the variable region of the second CH3 domain-containing polypeptide chain recognize different target epitopes, and the variable region of the third CH3 domain-containing polypeptide chain and the variable region of the fourth CH3 domain-containing polypeptide chain recognize the same target epitope.

[0390] 11. The method of claim 10, wherein the target epitope recognized by the variable region of the third CH3 domain-containing polypeptide chain and the variable region of the fourth CH3 domain-containing polypeptide chain is the same as the target epitope recognized by the variable region of the first CH3 domain-containing polypeptide chain or the variable region of the second CH3 domain-containing polypeptide chain.

[0391] 12. The method of claim 10, wherein the target epitope recognized by the variable region of the third CH3 domain-containing polypeptide chain and the variable region of the fourth CH3 domain-containing polypeptide chain is different from the target epitope recognized by the variable region of the first CH3 domain-containing polypeptide chain or the variable region of the second CH3 domain-containing polypeptide chain.

[0392] 13. The method of claim 8, wherein the variable region of the first CH3 domain-containing polypeptide chain and the variable region of the second CH3 domain-containing polypeptide chain recognize the same target epitope, and the variable region of the third CH3 domain-containing polypeptide chain and the variable region of the fourth CH3 domain-containing polypeptide chain recognize a second target epitope different from the target epitope recognized by the first variable region and the second variable region.

[0393] 14. The method of any one of items 8 to 13, wherein the target epitopes are located on the same target molecule.

[0394] 15. The method of claim 14, wherein the target molecule is a soluble molecule.

[0395] 16. The method of claim 14, wherein the target molecule is a membrane-bound molecule.

[0396] 17. The method of any one of items 8 to 13, wherein the target epitopes are located on different target molecules.

[0397] 18. The method of claim 17, wherein the different target molecules are expressed on the same cell.

[0398] 19. The method of claim 17, wherein the different target molecules are expressed on different cells.

[0399] 20. The method of claim 17, wherein the different target molecules are soluble molecules.

[0400] 21. The method of claim 17, wherein one target molecule is a soluble molecule and the second target molecule is a membrane-bound molecule.

[0401] 22. A method according to any one of items 1 to 21, wherein the at least two different immunoglobulin-like molecules are antibodies.

[0402] 23. The method of claim 1, wherein the means for preferential pairing comprises engineered complementary knob-into-hole mutations, disulfide bonds, charge mutations, or a combination thereof.

[0403] 24. The method of claim 23, wherein the means for preferential pairing is selected from Table B.

[0404] 25. The method of claim 1, wherein all four of the nucleic acid molecules have means for preferentially pairing the first CH3 domain-containing polypeptide with the second CH3 domain-containing polypeptide and the third CH3 domain-containing polypeptide with the fourth CH3 domain-containing polypeptide, wherein the means for preferentially pairing the first CH3 domain-containing polypeptide with the second CH3 domain-containing polypeptide are different from those for preferentially pairing the third CH3 domain-containing polypeptide with the fourth CH3 domain-containing polypeptide.

[0405] 26. The method of any one of items 8 to 13, 17 to 19 or 21, wherein at least one of the target epitopes is located on a tumor cell.

[0406] 27. The method of any one of items 8 to 13, 17 to 19 or 21, wherein at least one of the target epitopes is located on an effector cell.

[0407] 28. The method of claim 27, wherein the effector cells are NK cells, T cells, B cells, monocytes, macrophages, dendritic cells or neutrophils.

[0408] 29. The method of claim 27 or 28, wherein the target epitope is located on a CD3, CD16, CD25, CD28, CD64, CD89, NKG2D or NKp46 molecule.

[0409] 30. A mixture of at least two different immunoglobulin-like molecules obtainable by a method according to any one of items 1 to 29.

[0410] 31. A mixture according to item 30, wherein the at least two immunoglobulin-like molecules bind to different epitopes on the same antigen and / or different epitopes on different antigens.

[0411] 32. A mixture according to item 30 or 31, wherein the at least two different immunoglobulin-like molecules include at least one heterodimeric immunoglobulin-like molecule.

[0412] 33. A mixture according to any one of items 30 to 32, wherein two of said at least two different immunoglobulin-like molecules are heterodimeric immunoglobulin-like molecules.

[0413] 34. A recombinant host cell comprising nucleic acid sequences encoding at least a first CH3 domain-containing polypeptide chain, a second CH3 domain-containing polypeptide chain, a third CH3 domain-containing polypeptide chain, and a fourth CH3 domain-containing polypeptide chain, wherein at least two of the nucleic acid sequences have means for preferential pairing of the first CH3 domain-containing polypeptide with the second CH3 domain-containing polypeptide and of the third CH3 domain-containing polypeptide with the fourth CH3 domain-containing polypeptide.

[0414] 35. A recombinant host cell according to item 34, wherein the host cell also comprises a nucleic acid sequence encoding a common light chain.

[0415] 36. A pharmaceutical composition comprising at least two different immunoglobulin-like molecules according to any one of items 30 to 33 and a pharmaceutically acceptable carrier.

[0416] 37. A pharmaceutical composition according to item 36, wherein the at least two different immunoglobulin-like molecules are produced by a recombinant host cell according to item 34 or 35.

[0417] 38. A method for preparing a host cell for producing at least two different immunoglobulin-like molecules, the method comprising introducing into the host cell nucleic acid sequences encoding at least a first CH3 domain-containing polypeptide chain, a second CH3 domain-containing polypeptide chain, a third CH3 domain-containing polypeptide chain, and a fourth CH3 domain-containing polypeptide chain, wherein at least two of the nucleic acid sequences have means for preferential pairing of the first CH3 domain-containing polypeptide with the second CH3 domain-containing polypeptide and of the third CH3 domain-containing polypeptide with the fourth CH3 domain-containing polypeptide, wherein the nucleic acid sequences are introduced sequentially or simultaneously.

[0418] 39. The method according to item 38 further comprises the step of introducing a nucleic acid sequence encoding a common light chain into the host cell.

[0419] 40. A culture of the recombinant host cell of item 34 or 35 or a culture of the recombinant host cell obtained by the method according to item 38 or 39, which produces at least two different immunoglobulin-like molecules.

Claims

1. A method for producing at least two different monospecific immunoglobulin-like molecules from a single host cell, wherein each of the two monospecific immunoglobulin-like molecules comprises two CH3 domains capable of forming an interface, the method comprising providing in the cell: a first nucleic acid encoding a first CH3 domain-containing polypeptide chain, a second nucleic acid encoding a second CH3 domain-containing polypeptide chain, a third nucleic acid encoding a third CH3 domain-containing polypeptide chain, and a fourth nucleic acid encoding a fourth CH3 domain-containing polypeptide chain; wherein the first CH3 domain-containing polypeptide chain comprises an arginine (R) residue or a lysine (K) residue at amino acid position 399 and a glutamic acid (E) residue or an aspartic acid (D) residue at amino acid position 409 according to the EU numbering system; and wherein the second CH3 domain-containing polypeptide chain comprises an arginine (R) residue or a lysine (K) residue at amino acid position 399 and a glutamic acid (E) residue or an aspartic acid (D) residue at amino acid position 409 according to the EU numbering system; and wherein the CH3 domains of the third and fourth CH3 domain-containing polypeptide chains are wild-type; The method further comprises the steps of culturing the host cell and allowing the at least four nucleic acids to be expressed and harvesting the at least two different monospecific immunoglobulin-like molecules from the culture.

2. The method of claim 1, wherein the first CH3 domain-containing polypeptide chain and / or the second CH3 domain-containing polypeptide chain further comprises the substitution K392D or K392E according to the EU numbering system.

3. The method of claim 1 or 2, wherein the at least two different monospecific immunoglobulin-like molecules are at least two monospecific antibodies.

4. The method of claim 1 or 2, further comprising providing the host cell with a nucleic acid encoding a common light chain.

5. The method of claim 1 or 2, wherein each of the encoded CH3 domain-containing polypeptide chains or each of the encoded antibody heavy chains further comprises a variable region that recognizes a target epitope.

6. A mixture of at least two different monospecific immunoglobulin-like molecules or at least two different monospecific antibodies obtainable by a method according to any one of claims 1 to 5.

7. The mixture of claim 6, wherein the at least two different monospecific immunoglobulin-like molecules or the at least two different monospecific antibodies bind to different epitopes on the same antigen and / or different epitopes on different antigens.

8. A mixture of at least two different monospecific immunoglobulin-like molecules, wherein each of the two monospecific immunoglobulin-like molecules comprises two CH3 domains capable of forming an interface, wherein the two CH3 domains of the first immunoglobulin-like molecule comprise an arginine (R) residue or a lysine (K) residue at amino acid position 399 and a glutamic acid (E) residue or an aspartic acid (D) residue at amino acid position 409 according to the EU numbering system, and wherein the two CH3 domains of the second immunoglobulin-like molecule are wild-type.

9. A recombinant host cell comprising one or more nucleic acids encoding at least a first CH3 domain-containing polypeptide chain, a second CH3 domain-containing polypeptide chain, a third CH3 domain-containing polypeptide chain, and a fourth CH3 domain-containing polypeptide chain, wherein the first CH3 domain-containing polypeptide chain comprises an arginine (R) residue or a lysine (K) residue at amino acid position 399 and a glutamic acid (E) residue or an aspartic acid (D) residue at amino acid position 409 according to the EU numbering system, and wherein the second CH3 domain-containing polypeptide chain comprises an arginine (R) residue or a lysine (K) residue at amino acid position 399 and a glutamic acid (E) residue or an aspartic acid (D) residue at amino acid position 409 according to the EU numbering system, and wherein the CH3 domains of the third and fourth CH3 domain-containing polypeptide chains are wild-type.

10. The recombinant host cell of claim 9, wherein the first CH3 domain-containing polypeptide chain and / or the second CH3 domain-containing polypeptide chain further comprises the substitution K392D or K392E according to the EU numbering system.

11. The recombinant host cell of claim 9 or 10, wherein the host cell further comprises a nucleic acid encoding a common light chain.

12. A pharmaceutical composition comprising a mixture of at least two different monospecific immunoglobulin-like molecules or at least two different monospecific antibodies according to any one of claims 6 to 8 and a pharmaceutically acceptable carrier.

13. The pharmaceutical composition of claim 12, wherein the at least two different monospecific immunoglobulin-like molecules or at least two different monospecific antibodies are produced by the recombinant host cell according to any one of claims 9 to 11.

14. A method for preparing a host cell for producing at least two different monospecific immunoglobulin-like molecules, the method comprising introducing into the host cell one or more nucleic acids encoding at least a first CH3 domain-containing polypeptide chain, a second CH3 domain-containing polypeptide chain, a third CH3 domain-containing polypeptide chain, and a fourth CH3 domain-containing polypeptide chain, wherein the first CH3 domain-containing polypeptide chain comprises an arginine (R) residue or a lysine (K) residue at amino acid position 399 and a glutamic acid (E) residue or an aspartic acid (D) residue at amino acid position 409 according to the EU numbering system, and wherein the second CH3 domain-containing polypeptide chain comprises an arginine (R) residue or a lysine (K) residue at amino acid position 399 and a glutamic acid (E) residue or an aspartic acid (D) residue at amino acid position 409 according to the EU numbering system, and wherein the CH3 domains of the third and fourth CH3 domain-containing polypeptide chains are wild-type.

15. The method of claim 14, wherein the first CH3 domain-containing polypeptide chain and / or the second CH3 domain-containing polypeptide chain further comprises the substitution K392D or K392E according to the EU numbering system.

16. The method of claim 14 or 15, wherein the at least two different monospecific immunoglobulin-like molecules are antibodies.

17. The method of claim 14 or 15, further comprising the step of introducing into the host cell a nucleic acid encoding a common light chain.

18. A culture of the recombinant host cell of any one of claims 9 to 11 or a culture of the recombinant host cell obtained by the method of any one of claims 14 to 17, which produces at least two different monospecific immunoglobulin-like molecules.

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