Methods and means for the production of ig-like molecules

Abstract

To provide methods and means for the production of Ig-like molecules, relating to the fields of molecular biology, medicine and biological therapeutics, particularly the field of therapeutic antibodies for the treatment of various diseases.SOLUTION: The invention provides means and methods for producing one or more Ig-like molecules in a single host cell. Novel CH3 mutations enabling the production of monospecific and / or bispecific Ig-like molecules of interest are also provided.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the fields of molecular biology, medicine and biological therapeutics, in particular to the field of therapeutic antibodies used to treat a wide variety of diseases. [Background technology]

[0002] Currently, the most commonly used biologics are isolated human or humanized recombinant monoclonal antibodies that enhance the ability of the body's immune system to neutralize or eliminate cells and / or molecules involved in disease processes or eradicate invading pathogens or infectious agents.

[0003] Monoclonal antibodies bind to a specific site on an antigen, or epitope, and in therapeutic antibodies, the epitope is selected to perform a desired function, such as eliminating tumor cells, blocking receptor-ligand interactions, or neutralizing a virus.

[0004] Currently, there are about 30 monoclonal antibodies approved by the FDA, which are typically produced in large quantities and have their biophysical and biochemical properties thoroughly tested to ensure that each batch meets quality standards.

[0005] Despite these advantages, monoclonal antibodies have several disadvantages, some of which are related to their inherent monospecificity and the complexity of diseases.

[0006] Disease processes are often multifactorial in nature. The actions of disease-related factors may be redundant or synergistic, and crosstalk may occur between signaling networks due to increased expression of alternative receptors. Thus, blocking different disease-related factors or pathways may improve the efficacy of treatment. Due to the inherent monospecificity of monoclonal antibodies, they may only be able to interfere with one step in a complex disease process and may not be effective enough.

[0007] Apart from the issue of the multifaceted nature of disease processes, it has become clear that targeting a single epitope on a single cell, soluble protein, or pathogen may not be sufficient to treat the disease effectively, because the targeted epitope may no longer be available for the monoclonal antibody to bind and exert the desired effect. For example, tumor cells often evade monoclonal antibody treatment by down-regulating, mutating, or masking the target epitope on a growth factor receptor.

[0008] By activating other receptors and / or their ligands, tumor cells activate alternative pathways to continue growing and metastasizing. Similarly, viruses and other pathogens evade monoclonal antibody therapy by mutating, deleting, or masking their target epitopes.

[0009] Monoclonal antibodies that bind to a single epitope do not generate all of the effector mechanisms that polyclonal antibodies do, notably opsonization (making antigens more susceptible to phagocytosis), steric hindrance (antigens wrapped around antibodies are prevented from reaching host cells or mucosal surfaces), detoxification, agglutination, or precipitation of toxins (antibodies bound to some soluble antigens are agglutinated and subsequently removed), complement activation, and antibody-mediated cytotoxicity (antibodies cause target cell killing by NK cells and neutrophils).

[0010] Polyclonal antibodies for therapeutic applications can be obtained from pooled human serum. Such serum-derived therapeutic polyclonal antibodies can be used to treat or prevent viral infections such as rabies virus, cytomegalovirus and respiratory syncytial virus, to neutralize toxins such as tetanus toxin and botulinum toxin, or to prevent anti-D immunization.

[0011] The broader use of serum-derived polyclonal antibody preparations is hindered by the fact that the starting plasma can only be applied to a limited range of targets such as infectious diseases and toxins. Furthermore, the products are dependent on the donor blood supply, both in terms of quantity and suitability, resulting in considerable batch-to-batch variability. In addition, screening techniques have not kept pace with the constant evolution of viruses, and immunoglobulin preparations carry the risk of vectoring infectious disease transmission. Finally, the lengthy process of blood collection, screening, and immunoglobulin purification makes plasma-derived immunoglobulins costly to produce.

[0012] A mixture of monoclonal antibodies can enhance the efficacy of monoclonal antibodies while lacking the limitations associated with serum-derived polyclonal antibodies. In this field, combinations of two human or humanized monoclonal antibodies have been tested in preclinical models and clinical trials (e.g., a mixture of two monoclonal antibodies against the HER2 receptor, a mixture of two antibodies against the receptor EGFR, and two monoclonal antibodies against the rabies virus).

[0013] It has been shown in the field that the combination of two monoclonal antibodies can have additive or synergistic effects, inducing effector mechanisms that are not associated with either one of the antibodies alone. For example, a mixture of two monoclonal antibodies against EGFR or HER2 has been shown to more potently kill tumor cells. This is due to a combination of activities that includes enhanced receptor internalization and enhanced inhibition of downstream signaling pathways of the receptor, in addition to enhanced cytotoxicity by immune system effectors.

[0014] In combination therapy based on two monoclonal antibodies, the component antibodies are produced separately and then mixed at the protein level. The drawback of this approach is the enormous cost of developing clinical trials for the two antibodies separately and (partially) repeating the process for the combination. This can make therapy based on antibody combinations cost-prohibitive.

[0015] Alternatively, two recombinant cell lines producing the constituent monoclonal antibodies can be mixed in a fermenter and the resulting mixture of antibodies purified as a single sample (WO 2004 / 061104). The difficulty with this approach is that it is difficult to control the composition and therefore the reproducibility of the resulting recombinant polyclonal antibody preparation, especially when one considers that such composition may change over time as the cells are cultured.

[0016] In the last decade, bispecific antibodies have been developed, instead of using a combination of two antibodies, in which two different immunoglobulin molecules in a mixture bind to different epitopes on the same or different targets, whereas in bispecific antibodies this is achieved with a single immunoglobulin molecule.

[0017] By binding to two epitopes of the same or different targets, bispecific antibodies can exert effects similar to those of a combination of two antibodies binding to the same epitope. Furthermore, different effects have been observed due to the IgG format of bispecific antibodies combining two different monovalent binding sites in one molecule, and the mixture of two IgG antibodies combining two different bivalent binding molecules in one sample.

[0018] For this reason, from a technical and regulatory point of view, it is less laborious to develop a single bispecific antibody, since production, preclinical and clinical trials only require a single molecule. Thus, single bispecific antibody-based therapeutics are facilitated by a less laborious and cost-effective drug discovery process and provide more efficient antibody therapeutics.

[0019] Bispecific antibodies in the IgG format, consisting of two heavy chains and two light chains, can be produced in a variety of ways. For example, bispecific antibodies can be produced by fusing two cell lines secreting the antibodies or by expressing two antibodies in a single cell using recombinant DNA technology. These methods produce several types of antibodies, because the heavy chains corresponding to each antibody can form monospecific dimers (also called homodimers), which contain two identical heavy chain pairs with the same specificity, and bispecific dimers (also called heterodimers), which contain two different heavy chain pairs with different specificities.

[0020] Furthermore, the light and heavy chains from each antibody may pair randomly resulting in inappropriate, non-functional combinations. This problem, known as heavy and light chain mispairing, can be solved by selecting antibodies that share a common light chain to be expressed as bispecifics. However, even with a common light chain, expressing two heavy chains and one common light chain in a single cell results in three different antibody types: two monospecific 'parental' antibodies and a bispecific antibody. Therefore, the desired bispecific antibody needs to be generated from the resulting antibody mixture.

[0021] Several techniques have been employed to further increase the proportion of bispecific antibodies in mixtures of parental and bispecific antibodies and to reduce mispaired heavy and light chains, but there is a need for bispecific antibody formats that eliminate or minimize some of the problems mentioned above. Summary of the Invention [Problem to be solved by the invention]

[0022] In summary, the prior art provides various techniques and methods for producing monoclonal antibodies, bispecific antibodies, mixtures of monoclonal antibodies, or mixtures of monospecific and bispecific antibodies that can be applied in the treatment of patients.

[0023] However, as noted above, each of these current techniques and methods has its own drawbacks and limitations, and therefore there is a need to develop improved and / or alternative techniques for producing disease-modifying, multi-molecular targeted, mixture or bispecific approach-based biological therapeutics. [Means for solving the problem]

[0024] The present invention provides improved and / or alternative technology methods and means for producing disease modifying, molecular targeted, mixture or bispecific approach based biological therapeutics, and further provides the resulting products and uses of these methods and means.

[0025] In the prior art, various approaches have been described to promote the formation of specific bispecific antibodies of interest, which can reduce the composition of undesired antibodies in the resulting mixture.

[0026] For antibodies, CH3-CH3 interactions are known to play a major role in Fc dimerization (Ellerson JR., et al., J. Immunol 1976 (116) 510-517 and Deisenhofer J. biochemistry 1981 (20) 2361-2370). Furthermore, it is known that when two CH3 domains interact with each other, they face each other at a protein-protein interface consisting of "contact" residues (also called contact amino acids, interface residues or interface amino acids).

[0027] The contact amino acids of the first CH3 domain interact with one or more contact amino acids of the second CH3 domain. The contact amino acids are usually within 5.5 Å (preferably 4.5 Å) of each other in the three-dimensional structure of the antibody. The interaction between the contact residues of one CH3 domain and the contact residues of a different CH3 domain can be, for example, via 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 known forces.

[0028] It has been shown that approximately one-third of the contact amino acid side chains at the human IgG1 CH3 domain interface contribute to most of the folding and association of the domain, and it is anticipated that other (adjacent) amino acid residues may also affect interactions at this protein-protein interface.

[0029] In the prior art, approaches have been adopted to interfere with the dimerization of antibody heavy chains. Specific engineering of the CH3 domain has been applied to favor heterodimerization over homodimerization. Examples of such engineering of the CH3-CH3 interface are described, for example, in WO 1998 / 050431, Ridgeway et al., 1996, Merchant et al. 1998, also known as the Knob-into-Hole approach. This method introduces mutations that result in complementary protrusions and cavities.

[0030] Generally speaking, this method introduces protuberances at the contact surface of a first polypeptide and a corresponding cavity in a second polypeptide such that the protuberances are located in the cavity, thereby promoting the formation of heteromultimers and preventing the formation of homomultimers.

[0031] A "protuberance" or "knob" is created by replacing a short amino acid side chain at the interface of a first polypeptide with a longer amino acid side chain (e.g., tyrosine or tryptophan). A complementary "cavity" or "hole" of identical or similar size to the protuberance is created by replacing a long amino acid side chain at the interface of a second polypeptide with a shorter amino acid side chain (e.g., alanine or threonine). Protuberances and cavities can be created by synthetic means, such as modification of a nucleic acid encoding a polypeptide, or by synthesis of the peptide.

[0032] Using the knobs-into-hole technique alone, the proportion of the bispecific antibody of interest can be increased to 87% in a mixture of two parent antibodies and bispecific antibodies. Merchant et al. succeeded in increasing the proportion of the bispecific antibody in the mixture to 95% by introducing an additional disulfide bond between the two CH3 domains in the CH3-CH3 interface. Nevertheless, to use such bispecific antibodies as drugs, they must be purified from the homodimers and in a pharma- ceutical acceptable diluent or excipient. Purifying the heteromultimers from such mixtures is a major challenge due to the similar physicochemical properties of homodimers and heterodimers.

[0033] One of the objects of the present invention is to provide a method for producing bispecific antibodies from a single cell clone, which further improves the proportion of bispecific antibodies in the mixture. According to the present invention, the knob-into-hole technology can be used as one of the means, alone or in combination with other means, to achieve said further improved proportion of bispecific antibodies in the mixture.

[0034] Another example of such CH3-CH3 interface engineering is provided by heterodimeric Fc technology, which involves the design of bispecific and asymmetric fusion proteins through the introduction of strand-exchange engineered domain (SEED) CH3 heterodimers. These SEED·CH3 heterodimers are derivatives of human IgG and IgA composed of alternating segments of human IgA and IgG CH3 sequences. This results in a pair of complementary human SEED·CH3 heterodimers called SEED-bodies (Davis JH. et al., Protein Engineering, Design & Selection 2010(23)195-202; WO 2007 / 110205).

[0035] Yet another approach for producing the desired bispecific antibodies is based on electrostatic manipulation of naturally charged contact residues at the CH3-CH3 interface, as described, for example, in EP 01870459 or US 2010 / 0015133, WO 2007 / 147901, WO 2010 / 129304, Gunasekaran et al (2010) and WO 2009 / 089004.

[0036] The mutations in the heavy chain CH3 domain described in these publications replace naturally charged amino acid contact residues with amino acid residues of the opposite charge (charge reversal strategy), which changes the charge polarity at the opposing contact surfaces of the Fc dimer and can result in favorable attractive interactions when electrostatically compatible Fc chains are co-expressed, thus promoting the formation of the desired Fc heterodimers, while inhibiting the formation of undesirable Fc homodimers due to repulsive charge interactions.

[0037] At the CH3-CH3 interface, four characteristic pairs of charged residues have been reported to be involved in the interactions between the domains: D356 / K439', E357 / K370', K392 / D399' and D399 / K409' (following the numbering reported by Kabat (1991) where the residues of the first and second chains are separated by a " / " and the prime symbol (') represents the number of the residue in the second chain). Because the CH3-CH3 interface has two-fold symmetry, each characteristic pair of charged residues appears twice in native IgG (i.e., the electrostatic interactions of K439 / D356', K370 / E357', D399 / K392' and K409 / D399' are also present at the interface).

[0038] Taking advantage of this two-fold symmetry, it was shown that a single charge inversion, e.g., K409D in the first chain or D399'K in the second chain, reduced homodimer formation due to identical charge repulsion. This repulsion effect was further enhanced by inverting the opposite charge. Expression of different CH3 domains with different complementary charge inversions was shown to induce heterodimerization and, as a result, increase the proportion of bispecific species in the mixture.

[0039] The above-mentioned approach has allowed the percentage of bispecific antibodies produced from a single cell to be between about 76% and about 96%. One of the objects of the present invention is to provide a method for producing bispecific antibodies from a single cell with a further improved percentage of the desired bispecific antibody. According to the present invention, the electrostatic manipulation technique can be used as one of the means, alone or in combination with other means, such as the knob-into-hole approach, to achieve said further improved percentage of the desired (bispecific) antibody.

[0040] In one embodiment, the present invention provides a method for producing at least two different Ig-like molecules from a single host cell, each of the two Ig-like molecules comprising two CH3 domains capable of forming an interface, the method comprising providing to the cell: a. a first nucleic acid molecule encoding a polypeptide chain comprising a first CH3 domain, b. a second nucleic acid molecule encoding a polypeptide chain comprising a second CH3 domain, c. a third nucleic acid molecule encoding a polypeptide chain comprising a third CH3 domain, and d. a fourth nucleic acid molecule encoding a polypeptide chain comprising a fourth CH3 domain, wherein at least two of the nucleic acid molecules comprise means for selective pairing of a polypeptide comprising the first and second CH3 domains with a polypeptide comprising the third and fourth CH3 domains, the method further comprising the steps of culturing the host cell, expressing the at least four nucleic acid molecules, and recovering the at least two different Ig-like molecules from the culture.

[0041] For example, it is often desirable to produce multiple (bispecific) antibodies to more efficiently inhibit multiple biological pathways involved in a disease process or pathogen invasion, replication, and / or spread.

[0042] It is particularly useful to use a mixture of multiple bispecific antibodies for the treatment of a particular disease. For example, during treatment with antibodies or small molecule drugs, tumor cells use many different strategies to gain resistance. Resistance can involve multiple cell surface receptors and soluble molecules, and it would be beneficial to develop antibody-based cancer therapies that simultaneously address multiple such disease- and escape-related molecules.

[0043] When two or more such disease- and escape-related target molecules or epitopes are involved, a mixture of bispecific antibodies is a novel and interesting form of treatment. Such a mixture of bispecific antibodies is preferably produced from a single cell, facilitating the drug discovery process, making it less cumbersome from a regulatory point of view and more cost-effective and feasible from a pharmaceutical and clinical development point of view.

[0044] In a single cell based approach, it is desirable to use a method that allows for controlled and efficient production of bispecific antibodies, which can reduce or completely eliminate the need to separate the mixture of desired bispecific IgG molecules from undesired monospecific IgG molecules. In the prior art, monospecific and bispecific antibodies have been produced from single cells (WO 2004 / 009618). However, these mixtures are complex mixtures of several different bispecific and monospecific antibody types.

[0045] A further object of the present invention is to provide methods and means for producing a mixture of defined bispecific antibodies from a single cell. As described below, the methods preferably result in a (bispecific) antibody mixture with at least 95%, at least 97% or even higher than 99% percentage of dimeric IgG molecules, regardless of the amount of monomeric by-products. Generally speaking, in cells in which multiple, native IgG molecules are produced, half molecules (monomeric by-products) will be present but are easily removed by known size exclusion chromatography.

[0046] In one embodiment, the present invention provides a method for the production of a defined mixture containing at least two different Ig-like molecules in a single cell instead of one (bispecific) antibody of interest, with reduced or absent formation of undesired dimeric antibody species. The resulting mixture is well-defined and its composition is controlled by engineered mutations in the CH3 domain. Furthermore, modulating the expression levels and / or different transfection ratios used for expression influence the composition of the mixture.

[0047] In the method according to the invention, the CH3 domain encoded by the first nucleic acid molecule selectively pairs with the CH3 domain encoded by the second nucleic acid molecule, and the CH3 domain encoded by the third nucleic acid molecule selectively pairs with the CH3 domain encoded by the fourth nucleic acid molecule.Furthermore, the invention provides a mixture of at least two different Ig-like molecules obtainable by the method of the invention.

[0048] As used herein, "selective pairing of the polypeptides comprising the first and second CH3 domains" means that substantially all of the resulting dimers having a polypeptide comprising the first CH3 domain and / or a polypeptide comprising the second CH3 domain are dimers consisting of a pair of a polypeptide comprising one first CH3 domain and one polypeptide comprising the second CH3 domain.

[0049] Similarly, "selective pairing of the polypeptides comprising the third and fourth CH3 domains" means that substantially all of the resulting dimers having a polypeptide comprising a third CH3 domain and / or a polypeptide comprising a fourth CH3 domain are dimers consisting of a pair of a polypeptide comprising one third CH3 domain and one polypeptide comprising a fourth CH3 domain.

[0050] As a result, when a nucleic acid molecule encoding a polypeptide comprising four different (A, B, C, D) CH3 domains is introduced into a single cell, a mixture of primarily two specific Ig-like molecules is produced instead of a mixture of ten different Ig-like dimers (AA, AB, AC, AD, BB, BC, BD, CC, CD and DD).

[0051] As described in more detail below, in a preferred embodiment of the invention, the polypeptide chain comprising the first CH3 domain has the amino acid substitution T366K and the polypeptide chain comprising the second CH3 domain has the amino acid substitution L351D. These amino acid changes are preferred means for selective pairing of the polypeptide chains comprising the first and second CH3 domains.

[0052] The polypeptide chain comprising the first CH3 domain preferably further comprises the amino acid substitution L351K. The polypeptide chain comprising the second CH3 domain preferably further comprises an amino acid substitution selected from the group of Y349E, Y349D and L368E. Of this group, L368E is most preferred. In yet another preferred embodiment, the polypeptide chain comprising the third CH3 domain comprises the amino acid substitutions E356K and D399K, and the polypeptide chain comprising the fourth CH3 domain comprises the amino acid substitutions K392D and K409D.

[0053] In the method according to the present invention, each polypeptide chain comprising a CH3 domain preferably further comprises a variable region recognizing a target epitope.The variable regions that are part of the polypeptide chain comprising a CH3 domain preferably share a common light chain.In that case, only the VH of the variable regions differs, while the VL of all the variable regions are substantially identical.

[0054] Thus, in one preferred embodiment, the method according to the invention further comprises providing said host cell with a nucleic acid molecule encoding a common light chain. In one particularly preferred embodiment, each of the four variable regions of the polypeptide chain comprising four CH3 domains recognizes a different target epitope. For example, if the first nucleic acid molecule encodes a heavy chain further comprising a variable domain specific for antigen A, the second nucleic acid molecule encodes a heavy chain further comprising a variable domain specific for antigen B, the third nucleic acid molecule encodes a heavy chain further comprising a variable domain specific for antigen C, and the fourth nucleic acid molecule encodes a heavy chain further comprising a variable domain specific for antigen D. A mixture is then produced comprising bispecific Ig-like molecules specific for AB and bispecific Ig-like molecules specific for CD.

[0055] The formation of monospecific antibodies (AA, BB, CC or DD specific) or bispecific antibodies specific for AC, AD, BC or BD is reduced or eliminated by means of the selective pairing of the polypeptide comprising the first and second CH3 domains with the polypeptide comprising the third and fourth CH3 domains. For the production of a defined mixture containing more than two different Ig-like molecules, it is of course also possible to use additional nucleic acid molecules, e.g. encoding polypeptides comprising the fifth and sixth CH3 domains.

[0056] It is worth noting that the ratio of nucleic acids used in the method according to the invention does not have to be 1:1:1:1. Also, the ratio of the resulting expressed Ig-like molecules does not have to be 1:1. Using known means, it is possible to generate an antibody mixture with an optimized ratio. For example, the expression level of the nucleic acid molecules and thus the ratio of the resulting Ig-like molecules produced can be regulated by using different genetic elements such as promoters, enhancers and repressors, or by controlling the genomic integration site of the copy number of the DNA construct encoding the antibody.

[0057] The means for selective pairing preferably comprises complementary knob-into-hole mutations, disulfide bridges, charge mutations including charge reversal mutations, or combinations thereof. Those skilled in the art will appreciate that the means for selective pairing may be selected from a range of types of mutations. That is, at least four nucleic acid molecules encoding polypeptide chains comprising CH3 domains are required for charge mutations as means for selective pairing.

[0058] Furthermore, in certain cases, unengineered wild-type CH3 is also used for selective pairing of two polypeptide chains comprising wild-type CH3 domains. In a particularly preferred embodiment, said means for selective pairing comprises at least one CH3 mutation selected from Table B as described herein below.

[0059] A preferred embodiment provides a method according to the invention comprising, for all four of said nucleic acid molecules, means for selective pairing of a polypeptide comprising said first and second CH3 domains with a polypeptide comprising said third and fourth CH3 domains, wherein said means for selective pairing of a polypeptide comprising said first and second CH3 domains is different from the means for selective pairing of a polypeptide comprising said third and fourth CH3 domains.

[0060] An aspect of the invention provides a method according to the invention, wherein the means for selective pairing of the polypeptide comprising the first and second CH3 domains are different from the means for selective pairing of the polypeptide comprising the third and fourth CH3 domains. Different here means that the means for selective pairing of the polypeptide comprising the first and second CH3 domains are designed to favor selective pairing of the first and second chains, in such a way that interactions between the first and the polypeptide chain comprising the third and / or fourth CH3 domain are substantially prevented. In other words, dimerization of the polypeptide comprising the first CH3 domain with the third or fourth polypeptide is essentially prevented or nearly so. The polypeptide comprising the third and fourth CH3 domains is either wild type or is provided with means for selective pairing that are different from the means for selective pairing of the first and second CH3 domains.

[0061] Recent studies have focused on the production of single bispecific antibodies, for example using knobs-into-hole technology or mutation (inversion) of charged contact amino acids present in the CH3 domain. However, prior to the present invention, the production of a defined mixture of at least two (bispecific) Ig-like molecules without significant co-production of other dimeric by-products has not been feasible.

[0062] The present invention provides an efficient and controlled method for the production of mixtures of well-defined Ig-like molecules, including a high percentage of bispecifics in the mixture. In systems where two bispecifics are desired, a percentage of (two) bispecifics of at least 95%, at least 97% or higher can be obtained. This translates into only 5%, at most 3% or less of monospecific bivalent by-products. Of note, the amount of monomeric by-products (i.e., half molecules) is not critical, since these half molecules can be easily separated by exploiting their size differences.

[0063] In another preferred embodiment of the present invention, the variable regions of the polypeptide chains with the first and second CH3 domains recognize different target epitopes, while the variable regions of the polypeptide chains with the third and fourth CH3 domains recognize the same target epitope. This results mainly in one type of bispecific Ig-like molecule and one type of monospecific Ig-like molecule. For example, if the variable regions of the polypeptide chains with the first and second CH3 domains recognize different target epitopes, and if the variable regions of the polypeptide chains with the third and fourth CH3 domains recognize epitopes that are different from the target epitopes recognized by both the same first and second CH3 domains, a mixture of Ig-like molecules with specificity for AB or CC is created.

[0064] Further provided according to the invention is a method wherein the target epitopes recognized by the variable regions of the polypeptide chains comprising the third and fourth CH3 domains are identical but distinct from the target epitopes recognized by the variable regions of the polypeptide chains comprising the first or second CH3 domains.

[0065] In another method, when the variable regions of the polypeptide chains comprising the first and second CH3 domains recognize different target epitopes and the variable regions of the polypeptide chains comprising the third and fourth CH3 domains both recognize the same epitope as the polypeptide chain comprising the first or second CH3 domain, a mixture of Ig-like molecules with specificity for AB and AA, or AB and BB, is produced.

[0066] The method according to the invention provides that the target epitope recognized by the variable region of the polypeptide chain comprising the third and fourth CH3 domains is identical to the target epitope recognized by the variable region of the polypeptide chain comprising the first or second CH3 domain.

[0067] Another object of the present invention is to provide methods and means for producing defined mixtures of bispecific and monospecific antibodies in a single cell culture. A non-limiting example of such a well-defined mixture is a mixture of AB-specific bispecific and AA-specific monospecific antibodies. Another example is a mixture of AB-specific bispecific and BB-specific monospecific antibodies. Yet another example is a mixture of AB-specific bispecific and CC-specific monospecific antibodies. Again, suitable means and methods are provided for producing a mixture of desired antibodies having at least 90%, preferably 95%, most preferably at least 97% or even more than 99% of the desired antibodies.

[0068] In another embodiment of the method according to the invention, the variable regions of the polypeptide chains comprising the first and second CH3 domains recognize the same target epitope, while the variable region of the polypeptide chains comprising the third and fourth CH3 domains recognize a second target epitope different from the target epitopes recognized by the first and second variable regions. This results in the production of monospecific Ig-like molecules with predominantly AA or BB specificity. The formation of bispecific Ig-like molecules is reduced or eliminated.

[0069] In some 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 cross-linking of two identical target molecules is desired or when two targets are too far apart from each other to be bound by a single bispecific antibody. Producing a mixture of monospecific antibodies in a single cell can be advantageous as it can be considered as a single therapeutic product.

[0070] The therapeutic efficacy and safety of various monospecific antibodies have already been proven in the art, and they have been approved for production. Production of a mixture of monospecific antibodies in a single cell can facilitate testing of the efficacy and safety of some of these mixtures, and reduce the efficiency and cost of regulatory approval and production. However, a method for producing a specific mixture of monospecific antibodies in a single cell that can reduce the formation of bispecific by-products to less than 5% is currently not available. Another object of the present invention is to provide a means and method for producing such a mixture of well-defined homodimers that reduces the formation of bispecific antibodies to less than 5%.

[0071] The method according to the invention is suitable for the production of any desired mixture of bispecific and / or monospecific Ig-like molecules. Again, it is possible to use additional nucleic acid molecules encoding polypeptides comprising, for example, the fifth and sixth (and seventh and eighth, etc.) CH3 domains to produce defined mixtures containing more than two different Ig-like molecules.

[0072] Preferably, the method according to the present invention uses at least two CH3 domains that contain a combination of at least one of the mutations provided by the present invention.Through these mutations, a new specific interaction is formed between the two CH3 domains.These mutations according to the present invention are described in detail below.

[0073] As used herein, the term "Ig-like molecule" refers to a proteinaceous molecule having at least one immunoglobulin (Ig) domain. Said Ig-like molecule comprises a sequence having the function of at least one immunoglobulin CH3 domain, preferably the sequence comprises the CH3 domain of IgG1. A proteinaceous molecule having at least one CH3 domain can further comprise a specific binding moiety.

[0074] The CH3 domain of the present invention includes a means for selective pairing and is used for selective pairing of two proteinaceous molecules with CH3 domains to design a desired heterodimeric binding molecule or mixture of binding molecules. The binding moiety introduced into the proteinaceous molecule with CH3 domain can be any binding means, including those shown in the following non-limiting examples: single chain Fvs, single chain or tandem diabodies (TandAb®), VHH, Anticalins®, Nanobodies®, BiTE®, Fab, ankyrin repeat proteins or DARPINs®, Avimers®, DART, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins® or KALBITOR®.

[0075] 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 a polypeptide chain that comprises a CH3 domain preferably share a common light chain. In such a case, only the VH of the variable regions is different, while the VL of all variable regions is substantially identical.

[0076] Additionally or alternatively, cytokines, hormones, water-soluble ligands, receptors and / or peptides and other molecules can be introduced into the CH3 domain of the invention.

[0077] In a more preferred embodiment, the Ig-like molecule comprises a full length Fc main chain. In a most preferred embodiment, the Ig-like molecule is an antibody. Preferably, the variable regions of these antibodies share a common light chain but may differ in the VH region.

[0078] The term "antibody" as used herein refers to a proteinaceous molecule belonging to the immunoglobulin class of proteins, which contains a domain that binds to one or more epitopes on an antigen, the domain being derived from or having sequence homology to the variable region of an antibody. Known antibodies include several isotypes, such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM. The antibody according to the invention may be of any isotype or functional derivatives and / or fragments thereof. In a preferred embodiment, an antibody of the IgG isotype is produced as an Ig-like molecule, since IgG antibodies have a longer half-life compared to, for example, antibodies of other isotypes.

[0079] Antibodies produced by the methods of the invention can have sequences from any source, including murine and human sequences. Antibodies may be composed of sequences from one source, such as all from human antibodies, or may have sequences from two or more sources, resulting in what are called chimeric or humanized antibodies.

[0080] The closer the therapeutic antibody is to the subject's natural antibody, the better (e.g., a human antibody for a human subject). Antibody binding is expressed in terms of specificity and affinity. Specificity determines which antigen or epitope a binding domain will bind to. Affinity is a measure of the strength of binding to a particular antigen or epitope. Specific binding is defined as binding with an affinity (KD) of at least 1×10-5M, more preferably 1×10-7M, and even more preferably greater than 1×10-9M. Typically, therapeutic monoclonal antibodies are used with an affinity of 1×10-10M or higher.

[0081] The term "antigen" as used herein refers to a substance or molecule that, when introduced into the body, induces the immune system to produce antibodies. Antigens can come from a variety of sources, including pathogens, tumor or other abnormal cells, haptens, or autologous tissues, among others. At the molecular level, antigens are characterized by their ability to bind to the antigen-binding site of an antibody. Mixtures of antigens are also considered "antigens." That is, while tumor cell lysates or virus particles are often considered "antigens" by those skilled in the art, many antigenic determinants are present in such tumor cell lysates or preparations of virus particles.

[0082] An antigen comprises at least one, and often two or more, epitopes. The term "epitope" here refers to the part of an antigen that is recognized by the immune system, specifically antibodies, B cells, or T cells. Epitopes are usually thought of as derived from non-self proteins, although host-derived sequences can also be classified as epitopes.

[0083] The term "CH3 domain" is well known. The IgG structure has four chains, two light chains and two heavy chains. Each light chain has two domains, a variable region and a constant region (VL and CL). Each heavy chain has four domains, a variable region (VH) and three constant regions (CH1, CH2, CH3). The regions of the CH2 and CH3 domains of the heavy chains are called the Fc (Fragment crystallizable) portion, Fc fragment, Fc main chain or simply Fc.

[0084] IgG molecules are heterotetramers with two heavy chains and two light chains connected by disulfide bonds (-SS-) at the hinge. The heavy chains dimerize through interactions at the CH3-CH3 domain interface and at the hinge. The number of disulfide bonds in the hinge varies depending on the immunoglobulin subclass (Papadea and Check 1989).

[0085] The Fc fragment of an immunoglobulin is a dimer of two C-terminal constant regions, namely the CH2 and CH3 domains of the heavy chain. Part of its physiological function is the interaction with the complement system and with specific receptors on the surface of various cells. It is known that the interaction between the CH3 domains of two individual heavy chains plays an important role in inducing the dimerization of the heavy chains.

[0086] Thus, the CH3 domain plays a primary role in the association of the antibody heavy chains, and 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(20)2361-2370; Miller S., J. Mol. Biol. 1990(216)965-973; Padlan, Advances in Protein Chemistry 1996(49)57-133).

[0087] The variant CH3 domains of the invention can be used in conjunction with other antibody domains to generate bispecific or monospecific full-length antibodies. The specificity of the antibody as determined by the VH / VL combination generally does not affect the heavy chain dimerization behavior induced by the CH3 domain.

[0088] 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 the CH3 domain that may be involved in interdomain contact, which may be calculated by known techniques, including the calculation of the solvent accessible surface area (ASA) of residues in the CH3 domain in the presence and absence of a second chain, where residues that show a difference in ASA (>1 Å2) calculated under the two conditions are identified as contact residues (Lee and Richards J. Mol. Biol. 1971(55)379). Residues identified as contact residues are those 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, and 439 according to the EU numbering system (Table A).

[0089] [Table A]

[0090] The contact residues at the CH3-CH3 interface may be charged or neutral amino acid residues. As used herein, the term "charged amino acid residue" or "charged residue" refers to amino acid residues that have an electrically charged side chain. These may be positively charged side chains, such as those present in arginine (Arg, R), histidine (His, H) and lysine (Lys, K), or negatively charged side chains, such as those present in aspartic acid (Asp, D) and glutamic acid (Glu, E).

[0091] As used herein, the term "neutral amino acid residue" or neutral residue refers to all other amino acids that do not have an electrically 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).

[0092] As used herein, a "CH3-CH3 domain interface" or "CH3 interface", "CH3-CH3 pairing", "domain interface" or simply "interface" refers to the association of two CH3 domains of different polypeptides comprising CH3 domains as a result of an 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 an interaction may be, for example, van der Waals forces, hydrogen bonds, water-mediated hydrogen bonds, salt bridges or other electrostatic forces, attractive interactions between aromatic side chains, disulfide bond formation, or other known forces.

[0093] As used herein, said means of selective pairing of a polypeptide comprising a first and a second CH3 domain with a polypeptide comprising said third and fourth CH3 domain may be any means known in the art.

[0094] In one embodiment, at least one nucleic acid molecule encodes a CH3 domain containing large amino acid residues (i.e., "knobs" or "projections") at the contact residue positions, e.g., R, F, Y, W, I, or L, while at least one other nucleic acid molecule encodes a CH3 domain containing small amino acid residues (i.e., "holes" or "cavities") at the complementary contact residue positions, e.g., G, A, S, T, or V. The resulting CH3 domains are more likely to pair with each other due to the conformation of the contact amino acids. Here, the knob-into-hole technology has been described in detail above.

[0095] In a further embodiment of the invention, at least one nucleic acid molecule encodes a CH3 domain comprising an amino acid bearing an opposite charge compared to wild type at the contact residue position of the naturally charged residue, i.e., at the position originally K, H, R, D or E, while at least one other nucleic acid molecule encodes a CH3 domain comprising an amino acid bearing an opposite charge compared to wild type at the complementary contact residue position of the naturally charged residue. The resulting engineered CH3 domains are more likely to pair with each other due to the opposite charges of the contact amino acids, but are less likely to pair with identical CH3 domains due to electrostatic repulsion.

[0096] In one embodiment, the CH3 mutations described in EP 01870459, WO 2009 / 089004 and Gunasekaran et al (2010) are used.

[0097] In one embodiment, the means of selective pairing of the polypeptide comprising the first and second CH3 domains are "knob" and "hole" amino acid residues and the means of selective pairing of the polypeptide comprising the third and fourth CH3 domains are charge engineered amino acids. Preferably, the means of selective pairing of both the polypeptide comprising the first and second CH3 domains and the polypeptide comprising the third and fourth CH3 domains are charge engineered amino acids.

[0098] In one embodiment, the amino acid residues engineered for selective pairing of the polypeptides comprising the first and second CH3 domains are different compared to the amino acid residues engineered for selective pairing of the polypeptides comprising the third and fourth CH3 domains.

[0099] In a particularly preferred embodiment, at least the first and second nucleic acid molecules code for novel mutations of the CH3 domain provided by the present invention. As detailed below, the present invention provides novel CH3 mutations that allow the production of specific bispecific Ig-like molecules of interest without producing significant amounts of undesired (dimeric) by-products. Furthermore, the present invention provides novel CH3 mutations that allow the production of specific monospecific Ig-like molecules of interest without producing significant amounts of undesired (dimeric) by-products. Thus, the use of at least one of these CH3 mutations according to the present invention is preferred.

[0100] The terms "polypeptide", "polypeptide molecule" or "polypeptide chain" as used herein refer to a chain of amino acids covalently linked through peptide bonds. A protein is generally composed of one or more polypeptide molecules. In all polypeptides, one end, called the amino terminus, or N-terminus, has a free amino group. The other end, which has a free carboxyl group, is called the carboxyl terminus or C-terminus. Polypeptides according to the invention may be subject to a process of post-translational modification, such as glycosylation. Thus, a polypeptide chain comprising a CH3 domain according to the invention refers to a polypeptide chain comprising at least an Ig·CH3 domain, and may include those that have been post-translationally modified.

[0101] The term "nucleic acid molecule" as used herein is defined as a molecule comprised of a chain of nucleotides, more preferably DNA and / or RNA. In one embodiment, double-stranded RNA is used. In other embodiments, the nucleic acid molecules of the invention comprise other types of nucleic acid structures, such as, for example, DNA / RNA helices, peptide nucleic acid (PNA), locked nucleic acid (LNA) and / or ribozymes. Thus, the term "nucleic acid molecule" also encompasses chains that comprise non-natural nucleotides, modified nucleotides and / or non-nucleic acid components that exhibit the same function as natural nucleotides.

[0102] The invention further provides a method for making a host cell which produces at least two different Ig-like molecules, said method comprising the step of introducing into said host cell nucleic acid sequences encoding polypeptide chains comprising at least a first, a second, a third and a fourth CH3 domain, wherein at least two of said nucleic acid sequences provide a means for selective pairing of a polypeptide comprising said first and second CH3 domains with a polypeptide comprising said third and fourth CH3 domains, said nucleic acid sequences being introduced sequentially or simultaneously.

[0103] In a further aspect of the invention, a method of making a host cell for the production of a heterodimeric Ig-like molecule comprises introducing into the host cell a nucleic acid sequence encoding a polypeptide chain comprising at least a first and a second CH3 domain, wherein the polypeptide chain comprising the first CH3 domain comprises at least one substitution of a neutral amino acid residue with a positively charged amino acid residue and the polypeptide chain comprising the second CH3 domain comprises at least one substitution of a neutral amino acid residue with a negatively charged amino acid residue, the nucleic acid sequences being introduced one after the other or simultaneously. The method of making the host cell preferably comprises introducing into the host cell a nucleic acid sequence encoding a common light chain.

[0104] In one aspect further provided herein, in a recombinant host cell comprising a nucleic acid sequence encoding a polypeptide chain comprising at least a first, second, third and fourth CH3 domain, at least two of said nucleic acid molecules comprise means for selective pairing of a polypeptide comprising said first and second CH3 domains with a polypeptide comprising said third and fourth CH3 domains.

[0105] The present invention further provides a recombinant host cell comprising a nucleic acid sequence encoding a polypeptide chain comprising at least a first and a second CH3 domain, wherein the polypeptide chain comprising the first CH3 domain comprises at least one substitution of a neutral amino acid residue with a positively charged amino acid residue and the polypeptide chain comprising the second CH3 domain comprises at least one substitution of a neutral amino acid residue with a negatively charged amino acid residue.

[0106] A recombinant host cell according to the invention preferably comprises a nucleic acid sequence encoding a common light chain.

[0107] A "host cell" of the present invention may be any host cell capable of expressing a recombinant DNA molecule, including bacteria such as Escherichia (e.g., E. coli), Enterobacter, Salmonalla, Bacillus, Pseudomonas, Streptomyces, yeasts such as S. cerevisiae, K. lactis, P. pastoris, Candida, or Yarrowia, filamentous fungi such as Neurospora, Aspergillus oryzae, Aspergillus nidulans, and Aspergillus niger, insect cells such as Spodoptera frugiperda SF-9 or SF-21 cells, and preferably mouse cells, including CHO (Chinese hamster ovary) cells, BHK cells, SP2 / 0 cells, and NS-0 myeloma cells, primate cells such as COS and Vero cells, MDCK cells, BRL cells, and the like. These include mammalian cells such as 3A cells, hybridomas, tumor cells, immortalized primary cells, W138, HepG2, HeLa, HEK293, HT1080 or embryonic retinal cells such as PER.C6.

[0108] In choosing an expression system, mammalian cell expression vectors and hosts are often used to ensure that the antibody is appropriately glycosylated. Human cell lines, preferably PER.C6, are advantageously used to obtain antibodies that match the glycosylation pattern in humans. Conditions for growing or propagating cells (see Tissue Culture, Academic Press, Kruse and Paterson, editors (1973)) and conditions for expression of recombinant products may be somewhat different. Also, process optimization is usually performed to increase the product ratio and / or cell growth, respectively, by methods commonly known to those skilled in the art.

[0109] General guidelines, procedures and practical techniques for maximizing productivity in 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 literature (e.g., EP-A-0120694, EP-A-0314161, EP-A-0481790, EP-A-0523949, U.S. Pat. No. 4,816,567, WO 00 / 63403). Nucleic acid molecules encoding the light and heavy chains can be extrachromosomally copied and / or stably integrated into the host cell chromosome, the latter being preferred.

[0110] In a further aspect of the invention there is provided a culture of a recombinant host cell according to the invention or a culture of a recombinant host cell obtainable or obtainable by a method according to the invention, said culture producing at least either two different Ig-like molecules or a heterodimeric Ig-like molecule.

[0111] In obtaining expression of a sequence of a nucleic acid molecule encoding a polypeptide comprising a CH3 domain, the sequence capable of inducing such expression is known to be functionally related to the sequence of a nucleic acid molecule encoding a polypeptide comprising a CH3 domain. By functionally related, it is meant that the nucleic acid sequence encoding a polypeptide comprising a CH3 domain or a precursor thereof is related to the sequence capable of inducing expression in that it is capable of inducing expression of a polypeptide comprising a CH3 domain or a precursor thereof.

[0112] Useful expression vectors are already available, such as, for example, the Invitrogen line of commercial pcDNA vectors. When a sequence encoding a polypeptide of interest is properly inserted with respect to sequences that control the transcription and translation of the encoded polypeptide, the resulting expression cassette is useful for producing, or in other words, expressing, the polypeptide of interest.

[0113] Sequences that induce expression include promoters, enhancers, etc., and combinations thereof. They must be capable of functioning in the host cell, thereby inducing the expression of a functionally related nucleic acid sequence. Promoters can be constitutive or regulatable, and can be obtained from a variety of sources, including viral, prokaryotic or eukaryotic, or can be artificially designed.

[0114] Expression of the nucleic acid of interest can be driven by the native promoter or its derivatives, or by a completely heterologous promoter. Well-known and commonly used promoters for expression in eukaryotic cells include promoters derived from viruses such as adenovirus (e.g., the E1A promoter), promoters derived from cytomegalovirus (CMV) (e.g., the CMV immediate early (IE) promoter), promoters derived from simian virus 40 (SV40), etc. Suitable promoters can also be obtained from eukaryotic cells, including the metallothionein (MT) promoter, the elongation factor 1 alpha (EF-1α) promoter, the actin promoter, the immunoglobulin promoter, heat shock promoters, etc.

[0115] Any promoter or enhancer / promoter capable of inducing expression of a sequence of interest in a host cell is suitable for the present invention. In one embodiment, the sequence capable of inducing expression comprises a region of the CMV promoter, preferably comprising the region of nucleotides −735 to +95 of the CMV immediate early gene enhancer / promoter. As the skilled artisan will be aware, the expression sequence used in the present invention may preferably be a combination of elements that stabilize or enhance expression, such as insulators, matrix attachment regions, STAR elements (WO 03 / 004704), etc., which can increase the stability and / or level of expression.

[0116] The production of proteins in recombinant host cells has been extensively described, for example in Current Protocols in Protein Science, 1995, Coligan JE, Dunn BM, Ploegh HL, Speicher DW, Wingfield PT, ISBN 0-471-11184-8, Bendig, 1988. Culturing cells involves allowing the cells to metabolize and / or grow and / or divide and / or produce the protein of interest. This can be accomplished by any method known to those skilled in the art, including but not limited to feeding the cells. This can include growing attached to a surface, growing in suspension, or a combination of these.

[0117] Some culture conditions can be optimized by known methods to optimize the protein production. The culture can be, for example, in dishes, roller bottles or reactors, by batch, fed-batch, continuous or hollow fiber culture. For large-scale (continuous) recombinant protein production by cell culture, it is known that it is preferable to grow cells in suspension. It is also known that it is preferable to culture cells under conditions without animal or human serum or components of animal or human serum. Thus, the absence of additional animal or human proteins from the medium makes purification easier and safer. On the other hand, the synthetic medium is optimal in terms of reproducibility, making the system very reliable.

[0118] The Ig-like molecules are expressed in host cells and recovered from the cells or, preferably, from the cell culture medium by methods generally known to those skilled in the art. After recovery, these Ig-like molecules can be purified using known methods, including immunoprecipitation, centrifugation, filtration, size exclusion chromatography, affinity chromatography, cation and / or anion exchange chromatography, hydrophobic interaction chromatography, and the like. For mixtures of antibodies containing IgG molecules, Protein A or Protein G affinity chromatography can be suitably used (see, for example, U.S. Pat. No. 4,801,687 and U.S. Pat. No. 5,151,504).

[0119] The Ig-like molecules and / or mixtures thereof produced by the method according to the invention preferably have a common light chain. Thus, further provided is a method according to the invention further comprising providing said host cell with a nucleic acid molecule encoding a common light chain, which is a light chain capable of pairing with at least two different heavy chains, thereby forming a functional antigen-binding domain. A functional antigen-binding domain is capable of specifically binding to one antigen.

[0120] It is preferred to use a common light chain that can pair with all heavy chains produced by the method according to the invention. This avoids mispairing of incompatible heavy and light chains in the formation of a functional antigen-binding domain. In one embodiment, a common light chain of only one identical amino acid sequence can be used. In another embodiment, the skilled artisan will recognize that functionally equivalent light chains, even if they do not have identical amino acid sequences, are included in the meaning of "common". There are many variants of the light chain, which have mutations (deletions, substitutions, additions) that do not substantially affect the formation of a functional binding region. Thus, such variants can also bind to different heavy chains to form a functional antigen-binding domain.

[0121] The term "common light chain" as used herein refers to light chains that are identical or have differences in amino acid sequence, but where the resulting antibody retains binding specificity after pairing with a heavy chain. For example, it is possible to create or find light chains that are not identical but are still functionally equivalent by introducing and testing conservative amino acid changes and / or amino acid changes in regions that do not contribute or only partially contribute to binding specificity when paired with a heavy chain.

[0122] The term "common light chain" includes a combination of specific common light chains and such functionally equivalent variants. A detailed description of the use of common light chains is provided in WO 2004 / 009618. Preferably, the common light chain used in the present invention is a germline-like light chain, more preferably a germline-derived light chain, preferably a rearranged human germline-derived κ light chain, most preferably a rearranged human germline-derived κ light chain IgVκ1-39 / Jκ or IGVκ3-20 / Jκ.

[0123] Alternatively, instead of using a common light chain, and to avoid mispairing of incompatible heavy and light chains, one skilled in the art may choose to employ forced pairing of heavy and light chains, e.g., as described in WO 2009 / 080251, WO 2009 / 080252 and / or WO 2009 / 080253.

[0124] The present invention provides novel engineered CH3 domains as well as novel combinations of engineered CH3 mutations. Prior to the present invention, charged contact amino acids in the CH3 domain known to be involved in CH3-CH3 pairing were replaced with amino acids of the opposite charge (charge reversal), thereby affecting CH3-CH3 pairing.

[0125] The present invention mutations are an alternative to this approach because they substitute a charged residue for an amino acid in CH3 that is uncharged or neutral in the wild-type CH3. In this embodiment of the invention, rather than replacing a charged contact amino acid with an amino acid of the opposite charge, the uncharged CH3 amino acid is replaced with a charged one.

[0126] The approach of the present invention not only provides a method to efficiently drive the dimerization of the CH3 domains, but also has the advantage that at least one additional charge-charge interaction is created at the CH3 interface, which, in addition to the charge pair present at the CH3-CH3 interface, makes the dimers according to the present invention generally more stable compared to wild-type dimers (which are defined as bispecific IgG (AB) without CH3 engineering, in contrast to the parent homodimers (AA or BB)).

[0127] Surprisingly, it is also possible to further increase the percentage of one or more desired Ig-like molecules in the mixture. As mentioned above, known methods for preferentially producing bispecific antibodies generally produce undesirable dimeric by-products. For example, using knob-into-hole technology, the percentage of desired bispecific antibodies is at best 87%, whereas electrostatic engineering approaches, in which charged contact amino acids are replaced by amino acids of the opposite charge, result in a percentage of 96% (see, for example, Example 11).

[0128] Quite surprisingly, the inventors of the present invention have been able to introduce mutations that further increase the percentage of desired Ig-like molecules in the mixture. For example, in Example 17, it is shown that the method using the mutations according to the present invention results in such a high percentage of the desired bispecific antibodies that no dimeric by-products are detectable in the resulting mixture. Some unpaired half-molecules, in which only one heavy chain is paired with a common light chain, are present in the mixture, but these are the result of unequal expression of the heavy chains and can be easily separated from the mixture by size exclusion chromatography.

[0129] Thus, such mutations according to the invention allow the production of high percentages of bispecific Ig-like molecules in a single cell, substantially free of contaminating dimeric by-products, making them particularly suitable for pharmaceutical compositions.

[0130] A preferred embodiment of the present invention provides a method for producing a heterodimeric Ig-like molecule from a single cell, the Ig-like molecule comprising two CH3 domains capable of forming an interface, the method comprising the steps of: providing to the cell a. a first nucleic acid molecule encoding a polypeptide chain comprising a first CH3 domain; and b. a second nucleic acid molecule encoding a polypeptide chain comprising a second CH3 domain, the polypeptide chain comprising the first CH3 domain comprising at least one substitution of a neutral amino acid residue with a positively charged amino acid residue, and the polypeptide chain comprising the second CH3 domain comprising at least one substitution of a neutral amino acid residue with a negatively charged amino acid residue, the method further comprising the steps of culturing the host cell, expressing the two nucleic acid molecules, and recovering the heterodimeric Ig-like molecule from the culture. Preferably, the method further comprises the step of providing to the host cell a nucleic acid molecule encoding a common light chain, the advantages of which are outlined above.

[0131] The amino acid at position 366 in one CH3 domain and the amino acid at position 351 in the other CH3 domain have been reported to form a pair of contact residues at the CH3-CH3 interface, i.e., they are close enough in the three-dimensional conformation of the resulting Ig-like molecule that they can interact with each other. Thus, the first CH3 domain preferentially pairs with the second CH3 domain.

[0132] In one embodiment, the threonine (T) at position 366 of the first CH3 domain is replaced with a first charged amino acid and the leucine (L) at position 351 of the second CH3 domain is replaced with a second charged amino acid, said first and second charged amino acids having opposite charges. If a polypeptide comprising a first CH3 domain bearing a charged residue at position 366 further comprises a variable region with specificity for antigen A, and if a polypeptide comprising a second CH3 domain bearing a charged residue of the opposite charge at position 351 further comprises a variable region with specificity for antigen B, a bispecific Ig-like molecule with predominantly AB specificity is formed.

[0133] In a method further provided by the present invention, the means for selective pairing of a polypeptide comprising the first and second CH3 domains or the means for selective pairing of a polypeptide comprising the third and fourth CH3 domains is a substitution of a threonine at position 366 of the first or third CH3 domain with a first charged amino acid and a substitution of a 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.

[0134] One preferred combination of mutations according to the invention is a threonine (T) to lysine (K) substitution at position 366 in a polypeptide comprising a first CH3 domain and further comprising a variable region (e.g., specific for A), and a leucine (L) to aspartic acid (D) substitution at position 351 in a polypeptide comprising a second CH3 domain and further comprising a variable region (e.g., specific for B). This is denoted as the pair of mutations T366K / L351'D.

[0135] As mentioned above, it has been reported that the amino acids at position 366 of one CH3 domain and position 351 of the second CH3 domain are a pair of contact residues in the CH3-CH3 interface. The lysine introduced at position 366 and the aspartic acid introduced at position 351 have opposite charges, and these amino acids attract each other electrostatically. Thus, the first CH3 domain selectively attracts the second CH3 domain. Also, Ig-like molecules are predominantly formed by pairing the first CH3 domain with a lysine at position 366 and the second CH3 domain with an aspartic acid at position 351.

[0136] If a polypeptide comprising a first CH3 domain has specificity for antigen A and if a polypeptide comprising a second CH3 domain has specificity for antigen B, then an "AB" specific bispecific Ig-like molecule is predominantly formed. Of note, in some embodiments, the variable regions of the polypeptide chains comprising the first and second CH3 domains may both be identical, resulting in the formation of a monospecific Ig-like molecule (e.g., "AA" specificity).

[0137] As mentioned above, one advantage of the mutations according to the invention is that instead of replacing the original charged amino acid interaction, a novel interaction between the newly introduced pair of charged amino acids is generated, which has not been previously disclosed or suggested.

[0138] One aspect of the invention provides a method according to the invention for producing at least two different Ig-like molecules from a single host cell, wherein the polypeptide chain comprising the first CH3 domain comprises the amino acid substitution T366K and the polypeptide chain comprising the second CH3 domain comprises the amino acid substitution L351D.

[0139] One embodiment provides a method for producing a heterodimeric Ig-like molecule from a single cell, the Ig-like molecule comprising two CH3 domains capable of forming an interface, the method comprising providing to the cell - a first nucleic acid molecule encoding a polypeptide chain comprising a first CH3 domain, and - a second nucleic acid molecule encoding a polypeptide chain comprising a second CH3 domain, the polypeptide chain comprising the first CH3 domain comprising the amino acid substitution T366K and the polypeptide chain comprising the second CH3 domain comprising the amino acid substitution L351D, the method further comprising culturing the host cell, expressing the nucleic acid molecules and recovering the heterodimeric Ig-like molecule from the culture.

[0140] The above-described amino acid mutations according to the invention allow for the production of heterodimeric Ig-like molecules from a single cell, with less than 5% homodimer contamination, preferably less than 2%, more preferably less than 1%, or most preferably substantially free of homodimer contamination.

[0141] One embodiment provides a method for producing a heterodimeric Ig-like molecule from a single cell, said Ig-like molecule comprising two CH3 domains capable of forming an interface, with less than 5% presence of contaminating homodimers, preferably less than 2%, more preferably less than 1%, and most preferably substantially no homodimer contamination, said method comprising the steps of providing to said cell - a first nucleic acid molecule encoding a polypeptide chain comprising a first CH3 domain, and - a second nucleic acid molecule encoding a polypeptide chain comprising a second CH3 domain, said polypeptide chain comprising the first CH3 domain comprising the amino acid substitution T366K and said polypeptide chain comprising the second CH3 domain comprising the amino acid substitution L351D, said method further comprising the steps of culturing said host cell, expressing said two nucleic acid molecules, and recovering said heterodimeric Ig-like molecule from the culture.

[0142] Preferably, in the method of producing at least two different Ig-like molecules according to the invention or in the method of producing a heterodimeric Ig-like molecule according to the invention, said polypeptide chain comprising a first CH3 domain further comprises the amino acid substitution L351K. More preferably, said polypeptide chain comprising a second CH3 domain further comprises an amino acid substitution selected from the group consisting of Y349E, Y349D and L368E. Most preferably, said polypeptide chain comprising a second CH3 domain further comprises the amino acid substitution L368E.

[0143] Thus, in a preferred embodiment, the above-mentioned T366K / L351'D mutation according to the invention can be further combined with a leucine (L) to glutamic acid (E) substitution at position 368 of the second CH3 domain, which can for example be designated as a T366K / L351'D, L368'E mutation (but can also be designated in other ways such as T336K / L351D-L368E or T366K / L351D, L368E or T366K-L351D,L368E).

[0144] As shown in Example 17, introduction of this mutation into a polypeptide according to the invention with a first CH3 domain specific for antigen A and a second CH3 domain specific for antigen B allows the production of a particularly good percentage of bispecific Ig-like molecules with dual AB specificity. This pair of mutations allows the production of bispecific antibodies without the formation of any appreciable amount of homodimers.

[0145] A preferred embodiment provides a method for producing a heterodimeric Ig-like molecule from a single cell, said Ig-like molecule comprising two CH3 domains capable of forming an interface, wherein the presence of contaminating homodimers is less than 5%, preferably less than 2%, more preferably less than 1%, and most preferably substantially free of contaminating homodimers, said method comprising providing to said cell - a first nucleic acid molecule encoding a polypeptide chain comprising a first CH3 domain, and - a second nucleic acid molecule encoding a polypeptide chain comprising a second CH3 domain, wherein said polypeptide chain comprising the first CH3 domain comprises the amino acid substitution T366K and wherein said polypeptide chain comprising the second CH3 domain comprises the amino acid substitutions L351D and L368E, said method further comprising the steps of culturing said host cell, expressing said two nucleic acid molecules and recovering said heterodimeric Ig-like molecule from the culture.

[0146] In another preferred embodiment, the threonine (T) at position 366 in the first CH3 domain is substituted with a lysine (K), the leucine (L) at position 351 in the second CH3 domain is substituted with an aspartic acid (D) and the tyrosine (Y) at position 349 in said second CH3 domain is substituted with a glutamic acid (E), e.g., designated as a T366K / L351'D,Y349'E mutation, as well as, e.g., T366K-L351D:Y349E or T366K / L351D,Y349E or simply T366K / L351DY349E.

[0147] The Y349 residue is a neighboring residue of the residue at position 351 that may contribute to dimer interactions. In silico data indicate that Y349E not only leads to destabilization of the monodimer (higher calculated score) but also to stabilization of the heterodimer (lower calculated score), with glutamic acid (E) being more favored than aspartic acid (D) at position 349. Thus, introduction of a second amino acid substitution into a polypeptide with a second CH3 domain that already has an amino acid substitution at position 351 favors heterodimerization.

[0148] A particularly preferred embodiment provides a method for producing a heterodimeric Ig-like molecule from a single cell, said Ig-like molecule comprising two CH3 domains capable of forming an interface, and having less than 5% homodimer contamination, more preferably less than 2%, even more preferably less than 1% and most preferably substantially free, said method comprising providing to said cell - a first nucleic acid molecule encoding a polypeptide chain comprising a first CH3 domain, and - a second nucleic acid molecule encoding a polypeptide chain comprising a second CH3 domain, said polypeptide chain comprising the first CH3 domain comprising the amino acid substitution T366K and said polypeptide chain comprising the second CH3 domain comprising the amino acid substitutions L351D and Y349E, said method further comprising the steps of culturing said host cell, expressing said two nucleic acid molecules and recovering said heterodimeric Ig-like molecule from the culture.

[0149] In another preferred embodiment, the Threonine (T) at position 366 of the first CH3 domain is replaced by a Lysine (K), the Aspartic Acid (D) at position 351 of the second CH3 domain is replaced by a Leucine (L), the Tyrosine (Y) at position 349 of said second CH3 domain is replaced by a Glutamic Acid (E) and the Leucine (L) at position 368 of said second CH3 domain is replaced by a Glutamic Acid (E). This is denoted as T366K / L351'D,Y349'E,L368'E mutation. The two residues Y349 and L368 are residues that may contribute to dimer interactions.

[0150] In silico data show that Y349E and L368E not only destabilize the BB dimer (higher in silico score) but also stabilize the heterodimer (lower in silico score), with glutamic acid at positions 349 and 368 being preferred over aspartic acid (D). Thus, introducing a second and third amino acid substitution into the B-chain, which already has an amino acid substitution at position 351, further promotes heterodimer formation.

[0151] A particularly preferred embodiment provides a method for producing a heterodimeric Ig-like molecule from a single cell, said Ig-like molecule comprising two CH3 domains capable of forming an interface, and having less than 5% homodimer contamination, more preferably less than 2%, even more preferably less than 1% and most preferably substantially free, said method comprising the steps of providing to said cell - a first nucleic acid molecule encoding a polypeptide chain comprising a first CH3 domain, and - a second nucleic acid molecule encoding a polypeptide chain comprising a second CH3 domain, said polypeptide chain comprising the first CH3 domain comprising the amino acid substitution T366K and said polypeptide chain comprising the second CH3 domain comprising the amino acid substitutions L351D, Y349E and L368E, said method further comprising the steps of culturing said host cell, expressing said two nucleic acid molecules and recovering said heterodimeric Ig-like molecule from the culture.

[0152] In another preferred embodiment, the Threonine (T) at position 366 of the first CH3 domain is replaced by a Lysine (K), the Leucine (L) at position 351 of said first CH3 domain is replaced by a Lysine (K), the Leucine (L) at position 351 of said second CH3 domain is replaced by an Aspartic Acid (D) and the Leucine (L) at position 368 of said second CH3 domain is replaced by a Glutamic Acid (E). This is denoted as T366K,L351K / L351'D,L368'E mutation. As shown in the examples, this mutation also increases the percentage of the desired (bispecific) antibody. Moreover, this mutation allows to obtain bispecific antibodies without the formation of detectable amounts of homodimers.

[0153] Further provided is a method for producing a heterodimeric Ig-like molecule from a single cell, said Ig-like molecule comprising two CH3 domains capable of forming an interface, and having less than 5% homodimer contamination, more preferably less than 2%, even more preferably less than 1% and most preferably substantially free, said method comprising the steps of providing to said cell - a first nucleic acid molecule encoding a polypeptide chain comprising a first CH3 domain, and - a second nucleic acid molecule encoding a polypeptide chain comprising a second CH3 domain, said polypeptide chain comprising the first CH3 domain comprising amino acid substitutions T366K and L351K and said polypeptide chain comprising the second CH3 domain comprising amino acid substitutions L351D and L368E, said method further comprising the steps of culturing said host cell, expressing said two nucleic acid molecules and recovering said heterodimeric Ig-like molecule from the culture.

[0154] In another preferred embodiment, the Threonine (T) at position 366 in the first CH3 domain is replaced by a Lysine (K), the Leucine (L) at position 351 in the first CH3 domain is replaced by a Lysine (K), the Leucine (L) at position 351 in the second CH3 domain is replaced by an Aspartic Acid (D), the Tyrosine (Y) at position 349 in the second CH3 domain is replaced by an Aspartic Acid (D), and the Arginine (R) at position 355 in the second CH3 domain is replaced by an Aspartic Acid (D). This is denoted as the T366K,L351K / L351'D,Y349'D,R355'D mutation. This T366K-L351K / L351'D-Y349'D pair is further improved by the R355'D mutation in the B-chain, which improves the in silico score for BB and slightly improves the in silico score for AB.

[0155] Further provided is a method for producing a heterodimeric Ig-like molecule from a single cell, said Ig-like molecule comprising two CH3 domains capable of forming an interface, and wherein homodimer contamination is less than 5%, more preferably less than 2%, even more preferably less than 1% and most preferably substantially absent, said method comprising the steps of providing to said cell - a first nucleic acid molecule encoding a polypeptide chain comprising a first CH3 domain, and - a second nucleic acid molecule encoding a polypeptide chain comprising a second CH3 domain, said polypeptide chain comprising the first CH3 domain comprising amino acid substitutions T366K and L351K, and said polypeptide chain comprising the second CH3 domain comprising amino acid substitutions L351D, Y349D and R355D, said method further comprising the steps of culturing said host cell, expressing said two nucleic acid molecules and recovering said heterodimeric Ig-like molecule from the culture.

[0156] Table B lists mutations that are introduced into the CH3 domain as a preferred means of selective pairing for the generation of heterodimers or homodimers. [Table B]

[0157] A method according to the invention for the production of at least two different Ig-like molecules or a method according to the invention for the production of a heterodimeric Ig-like molecule is provided, whereby said means for selective pairing of polypeptides comprising said first and second CH3 domains and / or said means for selective pairing of polypeptides comprising said third and fourth CH3 domains comprise at least one combination of mutations as shown in Table B. Preferably, said means for selective pairing of polypeptides comprising said first and second CH3 domains and said means for selective pairing of polypeptides comprising said third and fourth CH3 domains comprise at least two combinations of mutations as shown in Table B.

[0158] The novel combination of CH3 mutations provided by the present invention allows the production of a mixture of at least two monospecific Ig-like molecules in a single cell, with less than 5%, preferably more than 2%, more preferably less than 1%, and most preferably substantially no bispecific Ig-like molecule contamination. These mutations according to the present invention are particularly suitable for the production of mixtures of monospecific antibodies. This is particularly useful when a high level of cross-linking of two identical target molecules is desired, when a sufficiently high density of antibodies on the target cell is required to activate a specific effector mechanism, such as complement-mediated lysis of tumor cells, or when the two targets are too far from each other to be bound by a single bispecific antibody, or to simplify the process of obtaining regulatory approval.

[0159] In such cases, it is often desirable to optimize the production platform for such monospecific antibodies. As shown in Example 10 and as inferred from the present invention, when a polypeptide comprising a first CH3 domain (e.g., having A specificity) is substituted for lysine (K) at position 392 with aspartic acid (D), a polypeptide comprising said first CH3 domain is substituted for aspartic acid (D) at position 399 with lysine (K), and a polypeptide comprising said first CH3 domain is substituted for lysine (K) at position 409 with aspartic acid (D), a mixture of at least two different monospecific Ig-like molecules, including a monospecific Ig-like molecule with AA specificity, can be produced in a single cell, with the formation of bispecific by-products (bispecific Ig-like molecules) being reduced to less than 5%, or less than 3%, or even to a level that is virtually not detectable at all.

[0160] Thus, the combination of the above mentioned mutations (designated K392D, D399K, K409D) is particularly preferred for the production of a mixture of monospecific Ig-like molecules. Those skilled in the art will recognize that functional mutants, i.e., K392E, D399R, K409E, can result in a similar effect. In addition, double mutants with substitutions of D399K and K409D, or K392D and K409D, D399R and K409E, etc., can also result in a similar effect.

[0161] The same applies to the combination of mutations where the glutamic acid (E) at position 356 in the polypeptide comprising the first CH3 domain is replaced by a lysine (K), the glutamic acid (E) at position 357 in the polypeptide comprising said first CH3 domain is replaced by a lysine (K), the lysine (K) at position 439 in the polypeptide comprising said first CH3 domain is replaced by an aspartic acid (D), and the lysine (K) at position 370 in the polypeptide comprising said first CH3 domain is replaced by an aspartic acid (D). This combination of mutations (designated E356K, E357K, K439D, K370D) is also particularly preferred for the production of mixtures of monospecific Ig-like molecules.

[0162] One skilled in the art will recognize that functional mutants, i.e., K356R, E357R, K439E, K370E, can result in similar effects. In addition, triple or double mutants with substitutions of E356K and K439D, E357K and K370D, or other functional mutants, can result in similar effects.

[0163] A further embodiment provides a method for producing at least two different monospecific Ig-like molecules from a single host cell, each of the two Ig-like molecules comprising two CH3 domains capable of forming an interface, the method comprising the steps of providing to the cell a first nucleic acid molecule encoding a polypeptide chain comprising a first CH3 domain having -A specificity and a second nucleic acid molecule encoding a polypeptide chain comprising a second CH3 domain having -B specificity, the polypeptide chain comprising the first CH3 domain comprising K392D, D399K and K409D mutations and the polypeptide chain comprising the second CH3 domain comprising a wild-type CH3 domain or comprising E356K, E357K, K439D and K370D mutations, the method further comprising culturing the host cell, expressing the nucleic acid molecules and recovering the at least two different Ig-like molecules from the culture.

[0164] Another embodiment provides a method for producing at least two different monospecific Ig-like molecules from a single host cell, each of the two Ig-like molecules comprising two CH3 domains capable of forming an interface, the method comprising the steps of providing to the cell a first nucleic acid molecule encoding a polypeptide chain comprising a first CH3 domain having -A specificity and a second nucleic acid molecule encoding a polypeptide chain comprising a second CH3 domain having -B specificity, the polypeptide chain comprising the first CH3 domain comprising a wild-type CH3 domain or comprising mutations K392D, D399K, K409D, and the polypeptide chain comprising the second CH3 domain comprising mutations E356K, E357K, K439D, K370D, the method further comprising culturing the host cell, expressing the nucleic acid molecules, and recovering the at least two different Ig-like molecules from the culture.

[0165] As shown in Example 10, two monospecific Ig-like molecules are produced in a single cell and the formation of bispecific Ig-like molecules is substantially undetectable. One skilled in the art can select a third nucleic acid molecule encoding a polypeptide chain with a wild-type or engineered CH3 domain and provide it to the host cell such that a mixture of three monospecific antibodies is produced.

[0166] In one embodiment of the present invention, there is provided a method for producing at least two different Ig-like molecules or a method for producing a heterodimeric Ig-like molecule according to the present invention, in which each polypeptide chain comprising a CH3 domain further comprises a variable region recognizing a different target epitope, and the target epitopes are located on the same molecule.

[0167] This allows for more efficient antagonism of the (biological) function of the target molecule compared to when only one epitope is targeted. For example, a heterodimeric Ig-like molecule can simultaneously bind to two epitopes present on a growth factor receptor or a soluble molecule important for tumor cell proliferation. This can effectively inhibit several independent signal transduction pathways, leading to uncontrolled proliferation. Any combination of at least two Ig-like molecules can simultaneously bind to two, three, or four epitopes present on such growth factor receptors or soluble molecules.

[0168] In one preferred embodiment, the target molecule is a water-soluble molecule, hi another preferred embodiment, the target molecule is a membrane-bound molecule.

[0169] In another embodiment of the invention, in a method for producing at least two different Ig-like molecules or a method for producing a heterodimeric Ig-like molecule according to the invention, each polypeptide chain comprising a CH3 domain further comprises a variable region recognizing a target epitope, the target epitope being located on a different molecule, in which case each different target molecule may be a water-soluble molecule or a membrane-bound molecule.

[0170] In one embodiment, the different target molecules are water-soluble molecules. Alternatively, one target molecule is a water-soluble molecule, while the second target molecule is a membrane-bound molecule. In yet another case, both target molecules are membrane-bound molecules. In one embodiment, the different target molecules are expressed in the same cell, while in another embodiment, the different target molecules are expressed in different cells.

[0171] As non-limiting examples, any heterodimeric Ig-like molecule or any combination of at least two Ig-like molecules is suitable for simultaneously inhibiting multiple membrane-bound receptors, simultaneously neutralizing multiple water-soluble molecules such as cytokines or growth factors to tumor cells, or neutralizing different viral serotypes or strains.

[0172] A preferred embodiment provides a method for producing at least two Ig-like molecules or heterodimeric Ig-like molecules according to the present invention, where 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 recruiting T cells or NK cells for killing tumor cells, for example. For example, at least one Ig-like molecule produced by the method according to the present invention can recruit immune effector cells, preferably human immune effector cells, by specifically binding to a target molecule located on immune effector cells.

[0173] In a further embodiment, the immune effector cells are activated after the Ig-like molecules bind to the target molecules. Induction of effector mechanisms includes, for example, redirection of immune-regulated cytotoxicity by the Ig-like molecules produced by the method according to the present invention. The Ig-like molecules can bind to molecules that cause cytotoxicity, such as T cell receptors or Fcγ receptors, thereby activating downstream immune effector pathways.

[0174] The term "immune effector cells" or "effector cells" as used herein refers to a repertoire of natural cell populations of the mammalian immune system that affect the viability of target cells upon activation. Immune effector cells include not only cells of the lymphoid lineage, such as natural killer (NK) cells, T cells, including cytotoxic T cells, or B cells, but also cells of the myeloid lineage, such as monocytes, macrophages, dendritic cells, and neutrophil granulocytes, are considered immune effector cells. Thus, the effector cells are preferably NK cells, T cells, B cells, monocytes, macrophages, dendritic cells, or neutrophil granulocytes.

[0175] Target antigens present on immune effector cells include CD3, CD16, CD25, CD28, CD64, CD89, NKG2D and NKp46. Further provided is a method for producing at least two different Ig-like molecules or a method for producing a heterodimeric Ig-like molecule according to the invention, wherein the target epitope is located on a CD3, CD16, CD25, CD28, CD64, CD89, NKG2D or NKp46 molecule. Target cell viability includes the cell's ability to survive, proliferate and / or interact with other cells.

[0176] One aspect of the present invention provides a method for producing a heterodimeric Ig-like molecule according to the present invention, wherein each polypeptide chain comprising a CH3 domain further comprises a variable region recognizing a target epitope. In one embodiment, the two variable regions of each polypeptide chain comprising a CH3 domain recognize the same target epitope with different affinities. In another embodiment, the two variable regions of each polypeptide chain comprising a CH3 domain recognize different target epitopes.

[0177] In other embodiments, the different target epitopes are located on the same target molecule, which can be a membrane-bound or water-soluble molecule. In other embodiments, the different target epitopes are located on different target molecules, which are expressed on the same cell or on different cells. Alternatively, the different target molecules can be water-soluble molecules, or one target molecule can be a water-soluble molecule and the second target molecule can be a membrane-bound molecule.

[0178] In a preferred embodiment, at least one target molecule of the heterodimeric Ig-like molecule is located on a tumor cell. In yet another preferred embodiment, at least one target molecule of the heterodimeric Ig-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 granulocyte, and said target epitope is located on a CD3, CD16, CD25, CD28, CD64, CD89, NKG2D or NKp46 molecule).

[0179] A preferred embodiment provides a method for producing at least two different Ig-like molecules or a method for producing a heterodimeric Ig-like molecule according to the present invention, wherein, as described above, said at least two different Ig-like molecules are antibodies, most preferably antibodies of the IgG isotype, even more preferably antibodies of the IgG1 isotype.

[0180] Further provided is an Ig-like molecule, a heterodimeric Ig-like molecule or a mixture of at least two Ig-like molecules obtainable by the method according to the invention, said (heterodimeric) Ig-like molecule or mixture of Ig-like molecules preferably comprising at least one CH3 mutation as set out in Table B. As well as a pharmaceutical composition comprising at least one Ig-like molecule or a mixture of at least two Ig-like molecules according to the invention, a (heterodimeric) Ig-like molecule or a mixture of at least two Ig-like molecules comprising at least one mutation as set out in Table B is also provided.

[0181] In one embodiment, the Ig-like molecule is a bispecific Ig-like molecule, such as a bispecific antibody. In another embodiment, the Ig-like molecule is a monospecific Ig-like molecule, such as a monospecific antibody. A preferred embodiment provides a mixture of at least two different Ig-like molecules obtainable by a method according to the invention, wherein the at least two different Ig-like molecules bind to different epitopes on the same antigen and / or different epitopes on different antigens.

[0182] Further provided are heterodimeric Ig-like molecules obtainable by the method according to the invention, said heterodimeric Ig-like molecules binding to different epitopes on the same antigen and / or different epitopes on different antigens. The advantages and preferred uses of the mixtures and antibodies are described above.

[0183] The present invention provides a mixture of at least two different Ig-like molecules obtainable by a method according to the invention, said at least two different Ig-like molecules comprising at least one heterodimeric Ig-like molecule. In one embodiment, two of said at least two different Ig-like molecules are heterodimeric Ig-like molecules.

[0184] Yet another preferred embodiment provides a heterodimeric antibody comprising two CH3 domains, one of which comprises the amino acid substitutions L351D and L368E and the other of which comprises the amino acid substitutions T366K and L351K, which are preferred means of selective pairing of the two CH3 domains as described above.

[0185] 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 the "DEKK combination mutation," "DEKK mutant," "DEKK pair," "DEKK engineered CH3 domain," "DEKK," or other names referring 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."

[0186] Also provided is a pharmaceutical composition comprising a (heterodimeric) Ig-like molecule or a mixture of at least two Ig-like molecules obtainable by any method according to the invention. The (heterodimeric) Ig-like molecule or the at least two Ig-like molecules according to the invention are preferably antibodies. The pharmaceutical composition comprises the (heterodimeric) Ig-like molecule, a mixture comprising monospecific or bispecific Ig-like molecules or a combination of monospecific and bispecific Ig-like molecules.

[0187] The pharmaceutical composition according to the present invention also comprises a pharma- ceutically acceptable carrier. As used herein, such a "pharma- ceutically acceptable carrier" refers to any and all solvents, salts, dispersion media, coatings, antibacterial and antifungal agents, isotonicity and absorption delaying agents, and similar physiologically compatible substances. Depending on the route of administration (e.g., intravenous, subcutaneous, intraarticular, etc.), the Ig-like molecule may be coated with a material to protect the Ig-like molecule from the action of acids and other natural conditions that may inactivate the Ig-like molecule.

[0188] In one aspect there is provided a pharmaceutical composition comprising a mixture of at least two Ig-like molecules obtainable by any method according to the invention, said at least two different Ig-like molecules being produced by a recombinant host cell according to the invention.Furthermore, there is provided a pharmaceutical composition comprising a heterodimeric Ig-like molecule obtainable by any method according to the invention, said heterodimeric Ig-like molecule being produced by a recombinant host cell according to the invention.

[0189] In addition to nucleic acid molecules encoding a polypeptide chain comprising a CH3 domain comprising at least one mutation described in Table B, recombinant host cells comprising at least one nucleic acid molecule encoding a polypeptide chain comprising a CH3 domain comprising at least one mutation described in Table B are also provided.

[0190] The present invention is further illustrated by the following examples, which are not intended to limit the invention and are presented merely for clarity of the invention. [Brief description of the drawings]

[0191] [Figure 1] FIG. 1A) is a schematic diagram of the construct vector MV1057. The stuffer region is the region into which the VH region of an antibody is cloned. FIG. 1B) is a schematic diagram of the phage display vector MV1043. [Diagram 2] This is the amino acid sequence of wild-type IgG1 Fc present in construct vector MV1057 (EU numbering system applied). [Diagram 3] Nucleotide and amino acid sequences of the VH region used for cloning into the various constructs. [Figure 4A] Mass spectrum data of transfection A. [Figure 4B] Mass spectrum data of transfection G. [Figure 4C] Mass spectrum data of transfection H. [Figure 5A] Mass spectrum data of transfection M. [Figure 5B] Mass spectrum data of transfection U. [Figure 6] Mass spectrum data of transfection O. [Figure 7A] Inhibition of homodimerization by substituting neutral amino acids with charged amino acids. [Figure 7B] Inhibition of homodimerization by substituting neutral amino acids with charged amino acids. [Figure 8] Figure 8(A) shows the native mass spectrometry (Native MS) spectrum of the transfected sample ZO(T366K / L351'D). Figure 8(B) shows the convoluted mass spectrometry spectrum of the transfected sample ZO(T366K / L351'D). The second / major peak is that of the bispecific molecule. [Figure 9] HADDOCK scores for experimentally validated mutation pairs. [Figure 10] Illustrated diagrams of CH3-CH3 interface interactions: FIG. 10A) for K409D:K392D / D399'K:E356'K, FIG. 10B) for D399K:E356K / D399'K:E356'K, and FIG. 10C) for K409D:K392D / K409'D:K392'D. [Figure 11] HADDOCK scores of various 366 / 351' charge variants. [Figure 12] Illustrated diagrams of CH3-CH3 interface interactions. Figure 12A) is for L351D / L351'D, and Figure 12B) is for L351D:S354A:R355D / L351'D:S354'A:R355'D. [Figure 13] HADDOCK scores for additional charge mutations near position L351. [Figure 14] HADDOCK scores for additional charge mutations near position T366 in strand A and position L351 in strand B. [Figure 15] A diagram of the CH3-CH3 interface interactions. [Figure 16] HADDOCK scores for variants near T366 / L351. [Figure 17] HADDOCK scores for additional variants near T366 / L351. [Figure 18]Example of nMS spectra of bispecific IgG obtained after co-expression of constructs T366K,L351K and L351D (left panel) or L351D,Y349E (right panel), scaled based on full-length monovalent IgG (half not shown). [Figure 19A] Mass spectrometry results showing the relative abundance of AA, AB, BB, A, and B (sum of all species is 100%). [Figure 19B] The data is the same as in FIG. 19A, but AB is omitted to make it easier to see the appearance of the undesirable AA, BB, A, and B. [Figure 20] Results of thermostability assay. Squares: wild type, triangles: charge-reversal pair E356K:D399K / K392D:K409D, circles: combinations of mutant CH3s shown above each graph. [Figure 21] Results of 10 freeze-thaw experiments. 1122 = first parent antibody BB; 1337 = second parent antibody AA; wild type = AA,AB,BB; CR = bispecific antibody with charge-reversed pair E356K:D399K / K392D:K409D; 3-6 and 9-12 = bispecific molecule combinations 3-6 and 9-12 in Table 15. [Figure 22] Serum stability was measured by ELISA using fibrinogen as the coated antigen. Figure 22A) ELISA data when IgG samples were diluted to 0.5 μg / ml. Figure 22B) ELISA data when IgG samples were diluted to 0.05 μg / ml. Results were normalized to 100% at T=0 (day). 1337 = second parent antibody AA; wild type = AA, AB, BB; CR = bispecific antibody with charge-reversed pair E356K:D399K / K392D:K409D; 3-6 and 9-12 = bispecific combinations 3-6 and 9-12 in Table 15. [Figure 23A] nMS results for experiments with transfection ratios from 1:5 to 5:1. Figure 23A shows the DEKK combination mutant, with "A" specificity on the DE side and "B" specificity on the KK side. [Figure 23B] nMS results for experiments with transfection ratios from 1:5 to 5:1. Figure 23B shows the DEKK combination mutants, with the "C" specificity on the DE side and the "B" specificity on the KK side. [Figure 23C] nMS results for experiments with transfection ratios ranging from 1:5 to 5:1. Figure 23C shows combinations of charge reversal mutations, with "A" specificity on the E356K:D399K side and "B" specificity on the K392D:K409D side. [Figure 24] nMS results for transfections #1-11 from Table 20. [Diagram 25] HADDOCK scores of dimers of different CH3 engineered vectors. Grey bars: desirable species AB and CD; Black bars: undesirable species AA, BB, CC, DD, AC, BC, AD, BD. [Figure 26] SDS-PAGE of transfections #1-11 from Table 20. Control samples DE / KK, DE / DE and KK / KK are included. [Figure 27A] nMS of transfection #9. [Figure 27B] nMS of transfection #11. [Figure 28A] This is nMS of gel filtered sample 1516:1516. [Figure 28B] This is nMS of gel filtered sample 1337:1337. [Figure 28C] This is the nMS of gel filtered sample 1516:1337. [Figure 29] Circulating concentrations of the DEKK engineered antibody and its two parent antibodies (pK study) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0192] Example 1: Amino acid substitutions generating a range of different CH3 domains To selectively promote or inhibit pairing of Ig-like molecules with CH3 domains and to obtain a wide variety of Ig-like molecules with different CH3 domains, we introduced a number of amino acid substitutions known to promote heterodimer formation, as well as a number of additional amino acid substitutions that have not previously been reported or tried but were selected to promote homodimer formation, into a construct vector (construct vector MV1057; Figure 1A).

[0193] Construct vector MV1057 comprises a nucleic acid sequence encoding the Fc portion of a normal wild-type IgG1 as depicted in FIG. 2. Table 1 lists the amino acid substitutions that were introduced into this wild-type Fc, resulting in a series of seven constructs. All constructs were made by Geneart. Constructs 1, 2 and 3, or alternatives thereof, have previously been reported to promote heterodimerization (EP 01870459, WO 2009 / 089004). Constructs 6 and 7 have also been reported (WO 98 / 50431). Constructs 4 and 5 are new and are designed to promote homodimerization.

[0194] [Table 1]

[0195] Example 2: Cloning VH into constructs with CH3 mutations Several antibody VH regions with known specificity and known binding ability to human IGKV1-39 light chains were used for cloning into these constructs. As mentioned above, all CH3 variants can be used with other antibody domains to generate full-length antibodies with bispecific or monospecificity. The antibody specificity defined by the VH / VL combination does not affect the heavy chain dimerization behavior induced by the CH3 domain. Model VH / VL combinations, where all light chains are based on human germline IGKV1-39 and VHs are varied, are used throughout this study.

[0196] Figure 3 shows the complete sequences and specificities of the antibody VH regions used throughout this study. The MF code is an internal designation for various VHs from Merus, for example, VH MF1337 has specificity for tetanus toxoid, MF1025 for porcine thyroglobulin, and MF1122 for bovine fibrinogen.

[0197] The VH region of the phage display vector MV1043 (Figure 1B) is cut with the restriction enzymes SfiI and BstEII (New England Biolabs / cat# R0123L and R0162L / , according to the manufacturer's instructions) to excise the VH fragment from the vector. The vector MV1057 is cut with SfiI and BstEII by standard methods (according to the manufacturer's instructions).

[0198] The fragment and vector are gel purified (Promega / cat# V3125 / according to manufacturer's instructions) and the cut vector and VH gene insert are isolated. Both are joined by ligation and the ligated nucleic acid is then transformed (according to manufacturer's instructions) into E. coli DH5α (Invitrogen / cat# 12297-016). The next day, single colonies are picked and sequenced to identify the vector with the appropriate insertion.

[0199] Example 3: Transfection and expression of full-length IgG in HEK293T cells Transfection of the various plasmids encoding the recloned VH variants as well as the common light chain human IGKV1-39 into HEK293T cells is performed using standard procedures (de Kruif et al Biotech Bioeng. 2010) to allow IgG expression. After transfection, the expression levels of IgG in the supernatant are measured with a ForteBIO Octet-QK system, which is based on Bio-Layer Interferometry (BLI) and allows real-time quantification and dynamic characterization of biomolecular interactions. For more information, see 1418274598331_0.com. If expression levels above 5 μg / ml are measured, IgG is purified using Protein A affinity purification.

[0200] Example 4: Purification of IgG Culture supernatants were purified using a Protein A column (GE Healthcare / cat# 11-0034-95 / according to manufacturer's instructions) and then eluted in 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 in PBS using a desalting column. Alternatively, IgG could be purified using Protein A beads (Sepharose beads CL-4B, GE healthcare cat#170780-01).

[0201] Example 5: Antigen-specific ELISA Antigen-specific ELISA is performed to assess the binding activity to the antigen. Antigen capture ELISA is performed to demonstrate the binding activity of the bispecific antibody. A biotinylated second antigen is used to detect the complex (de Kruif et al Biotech Bioeng. 2010).

[0202] (Example 6: SDS-PAGE) The purified IgG mixture was analyzed by SDS-PAGE (NuPAGE® 4-12% bis-tris gel / Invitrogen / cat# NP0323BOX) under reducing and non-reducing conditions by standard procedures, and the proteins in the gel were stained with colloidal blue reagent (PageBlue™ protein staining solution / Fermentas / cat# RO571).

[0203] Example 7: Enzymatic deglycosylation of IgG1 Since IgG glycosylation is heterogeneous, deglycosylation was performed to obtain a single product with a well-defined mass suitable for mass spectrometry analysis. One unit of N-glycosidase F (PNGase F; Roche Diagnostics, Mannheim, Germany) was added per 10 μg IgG1 and incubated overnight at 37°C. To remove the original purification buffer (0.1 M citrate buffer pH 3.0 / 1.0 M Tris-HCL pH 8.0), buffer exchange was performed using a 10 kDa MWCO centrifugal filter column (Millipore) and rebuffered in PBS. To remove the detached glycan chains, a similar buffer exchange procedure was performed and the buffer was exchanged into 150 mM ammonium acetate, pH 7.5. The filter was rinsed with 200 μl of 150 mM ammonium acetate, pH 7.5 for 12 min at 11,000 rpm and 4°C. After rinsing, 50 μl of deglycosylated IgG was applied to the filter and 450 μl of 150 mM ammonium acetate, pH 7.5 was added. This was followed by another centrifugation at 11,000 rpm for 12 min at 4°C. The centrifugation was repeated five times in total, each time adding fresh 150 mM pH 7.5 ammonium acetate buffer to a total volume of 500 μl. After the final centrifugation step, approximately 25 μl of the buffer-exchanged remaining deglycosylated IgG1 was collected, transferred to an Eppendorf tube, and prepared for mass spectrometry analysis.

[0204] Example 8: Native mass spectrometry Mass spectrometry is used to identify the different IgG species in the purified IgG mixture and to determine the ratios in which these IgG species are present. Briefly, 2-3 μl of 150 mM ammonium acetate, pH 7.5, containing 1 μM IgG was loaded onto a gold-plated borosilicate capillary tube (using a Sutter P-97 puller [Sutter Instruments Co., Novato, CA, USA] and an Edwards Scancoat six sputtering instrument [Edwards Laboratories, Milpitas, CA, USA]) and analyzed on an LCT1 mass analyzer (Waters Corp., Milford, MA, USA) tuned to optimize for high-mass detection (Tahallah et al., RCM 2001). A capillary voltage of 1300 V and a sampling cone voltage of 200 V were used; however, these settings were adjusted if higher resolution "signal-to-noise" ratios were required. To facilitate collisional cooling, the source backing pressure was increased to approximately 7.5 mbar. To measure denatured IgG1, the protein was nebulized at a concentration of 1 μM in 5% formic acid.

[0205] Example 9: Data processing and quantification The acquired spectra were processed using MassLynx 4.1 software (Waters Corp., Milford, MA, USA). Minimal smoothing was used and the spectra were centered. The mass of the species was calculated using each series of charge states. For each charge state, the corresponding intensity was assigned by MassLynx and added. This approach allows relative quantification for all species in a sample. Alternatively, peaks can be quantified using known area-under-the-curve (AUC) methods. All analyses were repeated three times to calculate the standard deviation of the IgG mass and its relative amount.

[0206] Example 10: Mixture of two or three monospecific antibodies from a single cell Several antibody VH regions (Figure 3) with known specificity and known human IGKV1-39 light chain binding capacity were used to reclone into wild type construct vector MV1057 or construct 4 or construct 5 of Table 1, resulting in vectors I-III (Table 2). The resulting vectors I, II, and III, each containing nucleic acid sequences encoding Ig heavy chains with different CH3 regions and different VH specificities and a common human light chain, were then transfected into cells. The transfections were performed alone to look for the formation of intact monospecific antibodies, or in combination with one or two other construct vectors to obtain a mixture of two or three monospecific antibodies. Table 3 lists the transfections and results.

[0207] [Table 2]

[0208] [Table 3]

[0209] Transfections A, G, and H yielded bivalent monospecific AA, BB, or CC homodimer formation only from cells transfected with either vector I, II, or III (Figure 4). For transfection A, this was predicted and previously demonstrated. Here, for the first time, we indeed report homodimerization of CH3-engineered Ig heavy chains containing the triple amino acid substitution construct 4 (i.e., K392D, D399K, K409D) or the quadruple amino acid substitution construct 5 (i.e., E356K, E357K, K439D, K370D) (transfections G and H).

[0210] Next, co-expression experiments of the two vectors in single cells were performed. Interestingly, co-expression of wild-type and CH3 engineered Ig heavy chains together with a common light chain in single cells, as shown by transfections M and N, resulted in a mixture of the two monospecific antibodies, without the presence of undesired bispecific antibodies, with only 4-5% of contaminating "other molecules" present in the mixture. "Other molecules" are defined as all molecules that do not have the mass of intact IgG, including half molecules consisting of a single heavy and light chain pair. Importantly, the "other" category does not include bispecific products.

[0211] In transfection M, the vector DNAs were transfected in equal proportions, resulting in a ratio of AA:BB of approximately 1:1. However, in transfection N, the ratio of AA:CC was approximately 10:1. Therefore, this transfection was repeated with adjusted DNA ratios (transfection U). In fact, when the vector DNA I:III was 1:5, the ratio of antibody products AA:CC in the mixture was approximately 1:1. Thus, as shown by transfections M and U, two different, substantially pure, monospecific antibodies can be expressed in a single cell without undesired by-products (i.e., no high presence of AC or half molecules A or C) (Figure 5). The novel CH3 modifications in constructs 4 and 5 are substantially different from the wild-type CH3 so that heterodimerization between wild-type and construct 4 or wild-type and construct 5 does not occur. This is advantageous for application in mass production of a mixture of monospecific antibodies from a single cell.

[0212] Similar to these results, two different CH3 engineered Ig heavy chains (constructs 4 and 5) are predicted to result in a mixture of only two different monospecific antibodies, without the presence of additional undesired species. If the CH3 modifications in construct 4 and construct 5 are substantially different, it is inferred that heterodimerization will not occur. In that case, co-expression of the CH3 engineered heavy chains of constructs 4 and 5 with the wild-type CH3 heavy chain in a single cell would result in only three monospecific antibodies.

[0213] Indeed, this result was observed. It was found that the expression of three different Ig heavy chains, designed to form homodimers rather than heterodimers, together with a common light chain in a single cell resulted in a mixture of three pure monospecific antibodies without any other contamination of the mixture (transfection O) (Figure 6). As can be clearly seen from Table 3, the ratio of antibodies AA:BB:CC was not 1:1:1, even when the same ratio of vector DNA was used in transfection O. Transfection with different ratios of vector DNA (1:1:10, transfection V) showed that the ratio of AA:BB:CC in the mixture could be manipulated to the desired ratio. Thus, as shown by these experiments, two or three substantially pure monospecific antibodies can be produced in a single cell without any undesired by-products. This is of great advantage for the mass production of mixtures of monospecific antibodies for therapeutic use.

[0214] Example 11: Mixture of two bispecific antibodies from a single cell The production of single bispecific antibodies with CH3 engineered heavy chains has been reported elsewhere. Here, this experiment was designed to investigate whether the production of a mixture of two different bispecific antibodies from a single cell is feasible. The VH regions of antibodies with known specificity and known binding capacity to human IGKV1-39 light chain (Figure 3) were recloned into vectors containing constructs 1-3 or 6-7 of Table 1, resulting in vectors IV-X (Table 4). Vectors IV-X, each containing a nucleic acid sequence encoding an Ig heavy chain with a different CH3 region and different VH specificity and a common human light chain, were then transfected into cells. The transfections were done either alone to demonstrate the prevention of the formation of intact monospecific antibodies, or in combination with other construct vectors to obtain bispecific antibodies or a mixture of two bispecific antibodies. Table 5 lists the transfections and results.

[0215] [Table 4]

[0216] [Table 5]

[0217] It has been shown that the CH3 engineered Ig heavy chains encoded by constructs 1 and 2 retain the ability to form homodimers when expressed in single cells (WO 2009 / 089004). However, a further report in WO 2009 / 089004 shows that CH3 domains engineered with triple charge pair mutations, such as construct 3, are no longer capable of homodimer formation when expressed alone. In the present study, these findings were only partially confirmed. In fact, transfections B, C and D showed the presence of full-length IgG in addition to a high proportion of unpaired half molecules, indicating homodimerization of the CH3 domains encoded by constructs 1 and 2. Transfections E and F also resulted in the production of full-length IgG in addition to unpaired half molecules, indicating that the triple charge mutation in construct 3 does not completely impair homodimerization. It was further shown that the “knob” and “hole” CH3 mutants of constructs 6 and 7 also formed homodimers (18% for “knob-knob” homodimers and 42% for “hole-hole” homodimers).

[0218] A CH3 mutant that completely inhibits homodimerization when expressed alone is preferred in order to prevent or minimize undesirable by-products (homodimers) when co-expressed with a second CH3 mutant for heterodimer formation.

[0219] Interestingly, this experiment showed for the first time that a mixture of bispecific antibodies could be expressed in a single cell, with virtually no homodimers in the mixture. In transfections K and L, the expected bispecific species BC+AB were indeed obtained (38%+47% in transfection K, 16%+60% in transfection L). A relatively high percentage of undesired half molecules was observed in both transfections (15% half molecule A+half molecule C in transfection K, 24% half molecule A+half molecule C in transfection L). The relatively high percentage of half molecules still present was due to low amounts of heavy chains in vector IV, due to unbalanced expression of the heavy chains of the matched pair. Therefore, in transfections S and T, the ratio of vector DNA was adjusted to 2:1:1 and transfections were performed again. This resulted in a pure bispecific IgG mixture with equal amounts of IgG heavy chains of the matched pair, no presence of IgG half molecules, and only 3% of homodimer BB. Ideally, this low percentage of monospecific product contamination should be reduced to essentially zero. It is therefore desirable to find additional CH3 mutants that would result in mixtures of bispecific antibodies that minimize the presence of contaminating monospecific antibodies.

[0220] This study shows, for the first time, that it is possible to produce a substantially pure mixture of two bispecific antibodies recognizing three different target epitopes in a single cell, while minimizing the presence of monospecific antibodies in the mixture.

[0221] Example 12: Various Mixtures Having shown that it is technically feasible to produce a mixture of two bispecific antibodies recognizing three epitopes, or a mixture of two or three monospecific antibodies from a single cell, we next explored the feasibility of the controlled production of various other mixtures.

[0222] A fourth antibody VH region with known specificity and known binding ability to human IGKV1-39 light chain was used to reclone into vectors containing constructs 1-3 or 7 in Table 1, resulting in vectors I', II', III', or X' (where ' indicates a different specificity compared to the corresponding vector number). Vectors I'-III', X' and IV-IX, each containing a nucleic acid sequence encoding an Ig heavy chain with a different CH3 region and different VH specificity and a common human light chain, were then transfected into cells. The transfections were done in combination with other construct vectors to obtain a mixture of different bispecific and / or monospecific antibodies. The different mixtures that can be obtained include a mixture of two bispecific antibodies recognizing four epitopes, two bispecific antibodies and one monospecific antibody, or one bispecific and one monospecific antibody from a single cell. Table 6 lists the transfections and the expected results.

[0223] [Table 6]

[0224] Although theoretically production of all mixtures is feasible, we know from other previous studies that large-scale production of previous knobs-into-holes mutants is hampered by stability issues, and therefore the mixtures resulting from transfections ZA, ZB, ZL, ZM, and ZN are expected to be problematic when moved to large-scale production.

[0225] In other words, the set of constructs in Table 1 may not be able to produce all theoretical mixtures in bulk from single cells, because knob-into-hole mutants are reported to be unstable, and it cannot be excluded that the CH3 domain with the "knob" or "hole" may dimerize with either the charged mutant or with the wild-type CH3 domain. Therefore, it is desirable to design new CH3 mutants for co-expression in single cells that are engineered to selectively form only homodimers or heterodimers and not homo- or heterodimerize with constructs 1-5 in Table 1.

[0226] Example 13: Identification of novel charge pair mutations The aim of this study was to engineer the CH3 region of IgG such that, when expressed in a single cell with a mixture of different IgG heavy chains, only heterodimers or homodimers would result. Here, the new engineered CH3 domain would not homodimerize or heterodimerize with known engineered or wild-type CH3 domains. Thus, as a first step in identifying new engineered CH3 domains that meet this criterion, a number of contact residues at the interface of the IgG CH3 domains were examined, either individually or in groups, with substitutions that would result in electrostatic repulsion between identical heavy chains, i.e., reduced homodimer formation. The aim was to obtain a list of residues that, when substituted with charged residues, would result in repulsion between identical heavy chains, such that these mutations could be used to induce homo- and / or heterodimer formation when different IgG heavy chains are expressed in a mixture. This would ensure that the resulting full-length IgG would be stable and produced at a high rate.

[0227] In further pursuit, the identified mutations are used to produce bispecific antibodies or mixtures of bispecific or monospecific antibodies by engineering matched pairs of CH3 residues in one or more IgG heavy chain-CH3 regions. Furthermore, the newly identified pairs of charge mutations are combined with existing pairs and used for expression in cells, so that multiple nucleic acid molecules encoding different heavy chains all have different and complementary CH3 mutations. This allows selectively obtaining mixtures of only monospecific antibodies or only bispecific antibodies, or mixtures of defined monospecific and bispecific antibodies. The residues tested in this study are contact residues previously identified (Deisenhofer J., 1981; Miller S., 1990; Padlan, 1996; Gunasekaran, 2010). The rationale for this approach is that repulsive charges are introduced to the contact residues of each available pair.

[0228] The samples are then analyzed by SDS-PAGE under non-reducing conditions to identify pairs that reduce dimer formation by looking for a band at approximately 72 kD. All resulting pairs are screened for single mutations or in combination with other single mutations, since it is unclear whether the repulsive electrostatic interactions of a single mismatched pair would be sufficient to obtain sufficient amounts of half-molecules for detection by this method. These mutations are also used in combination.

[0229] Amino acid substitutions were introduced into construct vector MV1057 by Geneart according to Table 7. Constructs were expressed by transfection into HEK293T cells according to standard procedures. IgG expression was measured by Octet. If production failed twice, the mutation was considered to be inhibitory to expression and was not pursued further.

[0230] [Table 7]

[0231] Supernatants containing ≥5 μg / ml IgG were analyzed by SDS-PAGE and purified with protein A. Proteins were stained with colloidal blue reagent. Homodimers were visualized as bands at approximately 150 kD. A smaller band at approximately 75 kD indicated the presence of half molecules (see negative controls: K392D, K409D). The blots are shown in Figure 7.

[0232] The results of SDS-PAGE were analyzed and scored, as shown in the rightmost column of Table 7. Many residues, including Q347, S354, Y349, L351, K360, T366, T394, and V397, are promising and should be further tested in combination. The selection here took into account both the high score in inhibiting homodimer formation and the availability of contact residues that can be modified without problems in relation to other non-complementary charges. For example, F405 and Y407 residues are known to have multiple interactions at the CH3-CH3 interface, including interactions with already charged residues. Among these interacting residues (see Table A), the introduction of multiple charge mutations can be problematic.

[0233] To test further combinatorial mutations, new constructs were made in vector MV1057 (Table 8) and antibody VH regions with known specificity and known binding ability to the human IGKV1-39 light chain were used to reclone into vectors containing these new constructs (see Table 9). Table 10 lists the transfections and results.

[0234] [Table 8]

[0235] [Table 9]

[0236] [Table 10]

[0237] Combinations of CH3 mutants were expressed and analyzed by SDS-PAGE (data not shown) and native mass spectrometry (MS). The results are summarized in Table 10. ZO transfection yielded the highest percentage of heterodimers in the mixture (69% AC). Interestingly, AA homodimers were absent in ZO transfection, while CC homodimers contained a small percentage (7%). Mass spectrometry showed that the remaining protein in the mixture consisted of half A molecules, which may be the result of unbalanced expression of A and C heavy chains. Raw MS data from transfection sample ZO are shown in Figure 8. Surprisingly, transfection ZO yielded a significant amount of bispecific product, whereas its opposite charge pair transfection ZP (L351K / T366'D vs. T366K / L351'D for ZO) did not yield similar results, with only 52% bispecific product observed and significant amounts of the two homodimers (30% AA and 13% CC). The explanation for this is that, because the negatively charged D is structurally very similar to T, the repulsion between T366D and itself is not strong enough, and therefore T366D can still form homodimers, which has indeed been observed.

[0238] It is expected that minor variants of the newly discovered T366K / L351'D pair (e.g., by testing all variants including the new constructs T366R and L351E) will result in a similar proportion of bispecific antibodies (BsAbs).

[0239] Example 14: Design of new CH3 mutations to induce efficient heterodimerization by HADDOCK As described in Example 13, the newly found charge pair T366K / L351'D increases the percentage of heterodimers in the mixture (69%) while also "contaminating" the mixture with a small percentage of undesired CC homodimers (7%) (L351D / L351'D) and a significant percentage of half molecule A (24%). In this example, an in silico approach is used to gain further insight into the amino acid residues involved in the interactions at the CH3 interface, test opposing complementary substitutions in the CH3 region, and find a novel CH3 pair containing complementary substitutions that inhibit efficient formation of homodimers of the two heavy chains while further increasing efficient heterodimerization.

[0240] HADDOCK (High Ambiguity Driven protein-protein DOCKing) is an information-driven, flexible docking approach for modeling biomolecular complexes. Unlike ab initio docking methods, HADDOCK encodes information about identified or predicted protein contacts in ambiguous interaction restraints (AIRs) to guide the docking process (de Vries et al., 2010). The input to the HADDOCK web server consists of protein structure files, such as crystal structures, NMR structure clusters, or structural models. After docking or fine-tuning, HADDOCK returns the so-called HADDOCK score, which is a weighted average of van der Waals energy, electrostatic energy, surface area covered, and desolvation energy. The HADDOCK score is often difficult to translate directly to experimental data, but is interpreted as an index of binding energy or affinity. In addition, HADDOCK provides structure files of the "top 4" structures resulting from the docking calculations that can be downloaded and visualized, allowing detailed analysis of the interactions of individual residues.

[0241] In this example, the interactions between the CH3 domains of the IgG1 heavy chain are studied. The Fc part of IgG (structure 1L6X), which has a high-resolution crystal structure, is used as the starting structure (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=1l6x; Idusogie, EE et al., JI 2000(164)4178-4184).

[0242] In Example 13, it was found that co-expression of vectors XIII and XVI resulted in the formation of contaminating CC homodimers (Table 10). HADDOCK was used to explore additional mutations in addition to T366K / L351'D that disrupt homodimerization.

[0243] The output of HADDOCK scoring is the calculated energies, the HADDOCK score (a weighted average of several energies), and four structure files corresponding to the four lowest energy structures found by the program. The HADDOCK score is used to compare different structures. The other energies are only used to get an indication of what is happening in the structure (e.g. better electrostatic interactions, less covered surface, higher van der Waals energy). The lower the HADDOCK score, the better. For each mutation pair, scores are calculated for AA, AB and BB dimers.

[0244] The set of mutation pairs from Example 12 was run in HADDOCK to see if the experimental data correlated with the calculated energies. All theoretical energies are shown in Table 11 and visualized in Figure 9.

[0245] [Table 11]

[0246] For the two wild-type CH3 domains, the HADDOCK scores are identical for AA, AB, and BB, since the CH3 regions of A and B are homotypic. In many other cases, the AB pair had the lowest score, as expected. For the T366K / L351D pair, the BB score was slightly better than the AB score (-210.6 vs. -212.5), but this difference is within the margin of error of the calculation. Using HADDOCK, the heterodimer structures of these pairs were visualized. For example, construct combinations 1-2, 1-1, and 2-2 are displayed in Figure 10. From these visualizations, it is clear that salt bridges are formed in the heterodimer (Figure 10A, left panel) and electrostatic repulsion occurs between residues of the same chain (Figure 10B and C, center and right panels). The higher HADDOCK score for the homodimer is explained by electrostatic repulsion of the mutated contact residues. These residues bend away from each other and do not interact with residues of the other chain, thus reducing affinity.

[0247] Table 11 and Figure 9 confirm the observations in Example 13. The AC heterodimer of T366K / L351'D and the CC homodimer of L351D / L351'D have similar energies, explaining the presence of both heterodimers and homodimers in the mixture. Meanwhile, the AA homodimer of T366K / T366'K is barely detectable in the mixture, and the T366K half molecule A is present. Table 11 and Figure 9 show that the HADDOCK score of the AA homodimer of T366K / T366'K is higher than that of the AC heterodimer. Thus, the formation of this homodimer is energetically less favorable.

[0248] Example 15: 366 / 351 Mutation In Example 13, it is hypothesized that alternative methods to the T366K / L351'D mutation charge pair can be designed to achieve similar results in terms of the proportion of bispecific antibodies in the mixture. The alternative methods can include substitutions of T366R, T366D, T366E, L351E, L351K, and L351R. The proportion of L351D / L351'D CC homodimers can be reduced by generating mutants with a 366 / 351 pair. All possible mutation pairs were run in HADDOCK, and the resulting scores are shown in Table 12 and visualized in Figure 11.

[0249] [Table 12]

[0250] Looking at the HADDOCK scores, it was observed that some mutations have a similar "pattern" when compared to T366K / L351'D. In many variants, AA homodimers were found to have higher HADDOCK scores than AB heterodimers, but BB homodimers were equally favored to AB heterodimers. The 351 residue is known to be "next door" to the same residue in the other chain; that is, the 351 residue in chain A pairs with the 351 residue in chain B at the CH3-CH3 interface. When the BB dimer forms, there is little negative effect of the same charge. Looking at the L351D / L351'D structure, the aspartates bend away from each other and have at least a stabilizing effect from the naturally occurring arginine at position 355, and further stabilization of the negative charge from the naturally occurring serine at position 354 (see Figure 12A). Mutating these residues (S354A and R355D) provides little improvement. From Figure 12B, it is clear that the main chain hydrogens of A354 are responsible for stabilizing the homodimer. From this series, the T366R / L351'E pair appears to be the most favorable with the lowest HADDOCK score for the bispecific molecule.

[0251] Example 16: Mutations around T366K / L351'D In this series of HADDOCK analyses, the starting structures were the T366K / L351'D or T366K / L351'E pairs. To identify additional mutations that could further improve the predicted value of the dual specificity rate of these A and B chains, mutations were added to the B chain and the HADDOCK scores and energies were calculated. When the CH3 domain structure was examined using a viewer for visualization of protein structures at the molecular level (YASARA, www.yasara.org), the distances between individual residues could be calculated. During the examination, two residues, Y349 and L368, were observed to be adjacent residues that could contribute positively or negatively to the dimer interactions. In this example, the effect of these mutations - in addition to the L351 mutation - on the dimer formation of homo- and heterodimers was examined (see FIG. 13). Both residues increase the stability of the heterodimer (lower HADDOCK score) and destabilize the BB dimer (higher HADDOCK score). Glutamic acids (E) at positions 349 and 368 were suggested to be preferred over aspartic acids (D). Thus, the introduction of a second amino acid mutation in the B chain, which already has an amino acid substitution at position 351, was suggested to favor heterodimerization.

[0252] In the next set of HADDOCK analyses, the T366K / L351'D pair was again taken as the starting structure. To the substitutions in the B chain (i.e., Y349D / E and L368E) that led to a further increase in heterodimerization, additional mutations were added to the A chain, which already contained the T366K substitution. As shown in Figure 14, there are several mutations that appear to favor the formation of bispecific heterodimers. In the T366K-L351K / L351'D-Y349'D pair, all four mutated residues are involved in the heterodimer pairing. 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 the latter heterodimer is -228.9, significantly lower than the -214.2 of T366K / L351'E-L368'E. This is explained by the hydrogen bond interaction at 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 increases the HADDOCK score of BB but also slightly increases the HADDOCK score of AB. Thus, when compared to the single mutation of T366K in the A chain, the additional L351K lowers the AB score and does not change the AA and BB scores much. Theoretically, this leads to a higher amount of bispecific heterodimers in the sample.

[0253] As is evident from Figure 11, it is speculated that changing position 366 to R instead of K is more effective in inducing heterodimerization. Therefore, some HADDOCK analyses shown in Figure 13 were repeated, this time with T366R instead of T366K in the A chain. Combining the double mutations R366 in the A chain and B chain was shown to be unfavorable (Figure 16). This is due to the large size of this residue, which interferes with other interface interactions even if all salt bridges with R366 are present in the structure. Furthermore, the HADDOCK score of the AA homodimer is lower for R366 than for K366. This also contributes unfavorably to the formation of heterodimers. Therefore, further HADDOCK analyses with R366 at the interface were discontinued.

[0254] A total of 14 best performing pairs were selected from HADDOCK predictions (see Table 13 and Figure 17). Some pairs contain the R355D substitution to remove the stabilizing effect of the naturally occurring R355 on the L351 / L351'D interaction.

[0255] [Table 13]

[0256] Example 17: In vitro expression of bispecific molecules using CH3 mutations based on HADDOCK predictions As suggested by the analysis in Example 16, some CH3 mutants with additional mutations around the T366K / L351'D pair can produce mixtures with a higher percentage of bispecific components and a lower percentage of homodimeric components. These best performing pairs were selected for production and further analysis. In addition, constructs T366R and L351E were also generated. A list of the constructs that were generated and used to reclone antibody VH regions with known specificity and binding ability to the human IGKV1-39 light chain is shown in Table 14.

[0257] The expression levels of IgG containing individual constructs reported in Example 13 above were repeated for the constructs listed in Table 14. The aim was to assess which constructs would homodimerize in the absence of a compatible partner for heterodimerization. Ideally, a high percentage of half molecules and a low percentage of homodimers would be formed. As controls, constructs containing previously reported charge mutations and constructs containing previously reported knob-in-hole mutations were also used for expression of whole IgG in recombinant cells. Protein A purified supernatants were analyzed by SDS-PAGE and the results, along with scores, are shown in Table 14.

[0258] [Table 14]

[0259] 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 the amino acid substitutions of the previous constructs are shown in Table 15. Expression of two different heavy chains with the amino acid substitutions T366K and L351'D:L368'E, respectively, allowed to obtain about 87% bispecific heterodimers AB in the mixture, without the presence of homodimers AA or BB (combination number 3 in Table 15). About 12% of half molecules containing the T366K substitution (half molecule A) were observed. Furthermore, it was found that the percentage of bispecific heterodimers AB increased when the additional amino acid substitution L351K was introduced in the first heavy chain. For example, co-expression of two different heavy chains with the amino acid substitutions T366K:L351K and L351'D:L368'E, respectively, resulted in approximately 92% bispecific heterodimer AB, while AA and BB homodimers were virtually absent from the mixture (combination number 12 in Table 15).

[0260] The combination of 10 and 11 also resulted in a favorable distribution, with a high percentage of heterodimers and virtually no homodimers. The absence of homodimers is advantageous because the fraction containing intact IgG molecules is composed only of heterodimers AB. For purification and subsequent therapeutic applications, half molecules can be removed by standard approaches such as size-exclusion chromatography. The known charge mutants and knob-into-hole mutants do not remove the "contaminating" homodimeric antibodies. However, it would be advantageous to apply these newly identified charge mutants to the bispecific antibody production process.

[0261] In addition, the T366K / L351'D:L368'E and T366K:L351K / L351'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: whereas the previously described charge variants are based on a reversal of the original charge within the CH3-CH3 interface, the newly identified charge variants add an additional charge pair (charge-charge interaction) at the CH3-CH3 interface. The introduction of an additional charge pair at the CH3-CH3 interface further increases the stability of the interface and thus the stability of the intact antibody. The same is true for the mutations used in combination numbers 4, 5, 6, 9, 10, and 11. This leads to a favorable proportion of bispecific heterodimers with very low proportions of AA and BB homodimers in the mixture.

[0262] [Table 15]

[0263] (Native MS) Native MS was performed on all dual specificity samples. The resulting graphs were analyzed to derive the relative ratios of the species of interest in two ways: peak height and peak area. The peak area method is the more scientifically correct analysis method, but since all analyses in other studies to date have been done with peak height, both methods were included in the analysis for comparison. The difference between the two methods was within the error range of the measurement, so only the peak area values ​​were used for subsequent measurements.

[0264] Two typical spectra are shown in Figure 18. A graphical summary of the results is shown in Figure 19. Values ​​can be found in Table 15. In about half of the samples the total contamination with monospecific IgG was below 5% and in only three cases it exceeded 10%, whereas with wild type IgG about 50% monospecific IgG would be expected to be found in the mixture.

[0265] Ten combinations of two different heavy chains were selected for further analysis from Table 15. These 10 combinations include combinations 1, 2, 3, 4, 5, 6, 9, 10, 11 and 12 (Table 15). The selection of these 10 was based not only on the low percentage of homodimers present in the mixtures derived by nMS, but also on the overall physicochemical properties including product yield, SDS-PAGE and number of mutations in the CH3 domain.

[0266] Example 18: Analysis of IgG stability In this study, we further analyze the stability of the Fc portion of IgG molecules for a series of CH3 mutation pairs that yield a high percentage of bispecific heterodimers in the intact IgG fraction and very low amounts (<5%) of the parental IgG. The mutated CH3 domains used to promote heavy chain heterodimerization can have unexpected destabilizing effects on the Fc region of IgG. This can result in undesirable properties such as reduced in vivo half-life, reduced effector functions and / or increased immunogenicity.

[0267] The newly identified charge pairs were compared to wild-type bispecific molecules and to bispecific molecules containing previously identified charge mutations (Chain A with Construct 1 and Chain B with Construct 2). All bispecific molecules in this study contain identical heavy and light chain variable regions, ensuring that any observed effects are due to mutations in the Fc portion of the molecules and not due to differences in the variable regions.

[0268] A series of stability studies will be performed on these bispecific molecules, including spectroscopic (UV-Vis light absorption, fluorescence and light scattering) and microscopic (optical and fluorescence microscopy with Nile Red staining) analyses that provide information on the aggregation state of the CH3 variants.

[0269] UV-Vis absorption spectra were recorded at 25°C on a double-beam, two-monochromator Cary 300 Bio spectrophotometer. Spectra were monitored between 250 and 400 nm with a 1 cm path length. Absorbance at 320 nm and longer wavelengths provides information on the aggregation state of IgG.

[0270] Intrinsic fluorescence spectra are monitored at 25° C. using a FluoroMax fluorometer. Fluorescence methods are optimized accordingly. Fluorescence emission provides information about conformation and aggregation properties.

[0271] 90° light scattering spectra are monitored at 25°C using a FluoroMax fluorometer running a synchronous scan (λem=λex) from 400 nm to 750 nm with an integration time of 0.01 seconds. The excitation and emission slits are optimized accordingly. For example, right angle light scattering can distinguish the presence of 5% dimers in an IgG sample.

[0272] For fluorescence microscopy with Nile red staining, Nile red in ethanol is added to the sample immediately before measurement. The sample is loaded onto a microscope slide and analyzed by fluorescence microscopy. Particles are counted; the smallest particle size observed by fluorescence microscopy is approximately 0.5 μm.

[0273] Stresses on proteins such as temperature, pH, mechanical stress or denaturants can lead to conformational changes (e.g. unfolding) and / or aggregation. Previously, charge engineered bispecific antibodies have been reported to have lower melting temperatures of modified CH3 (Gunasekaran 2010). Thus, these studies aim to distinguish the novel charge variants of the present invention from existing and known charge variants.

[0274] Thermostability studies were performed with the Octet using Protein A biosensor and FcRn to IgG. To investigate the thermostability of CH3 engineered IgG, samples were incubated at 4, 50, 55, 60, 65, 70 and 75°C for 1 h at a concentration of 100 μg / ml (PBS in a PCR machine). After this, samples were slowly cooled to 25°C in 15 min and kept at this temperature for 2 h before being stored at 4°C until the next day. Precipitated antibodies were removed by centrifugation and the total IgG concentration of water-soluble antibodies was derived with the Octet using the Octet Protein A biosensor (1 / 10 PBS dilution).

[0275] Assays measuring binding of CH3 engineered IgG to FcRn are being investigated using Octet. Protein L biosensors are used to bind IgG light chains to the sensor and then incubated with FcRn in solution. Alternatively, Anti-Penta-HIS biosensors can be used to bind His-tagged FcRn proteins and incubated with the IgG of interest. These methods may be more sensitive than Protein A biosensors and may be used for thermal stability studies.

[0276] All samples are also subjected to serum stability analysis. Briefly, (engineered) IgG samples are incubated in human serum at 37°C, while control samples are kept at 4°C. After 1, 2, 3 and 4 weeks, samples are centrifuged and precipitated IgG is removed. Samples are then spiked in an antigen-specific ELISA to determine the relative amount of functional IgG. A purified control antibody is acutely dosed in human serum and used as a reference group.

[0277] Example 19: Stability analysis In previous experiments, co-expression of two different heavy chains with CH3 mutations and a common light chain resulted in a high percentage of bispecific antibodies (Example 17).

[0278] Eight combinations of two different heavy chains were selected from Table 15 for further analysis. These eight combinations included combinations 3, 4, 5, 6, 9, 10, 11 and 12 (Table 15). In this study, these eight combinations were analyzed, focusing on the stability of the Fc portion of IgG. Control groups included wild-type bispecific molecules (i.e., no CH3 mutations) and / or previously reported CH3 charge mutations. Of note, for wild-type bispecific molecules, the two heavy chains and a common light chain were co-expressed without any means of selectively inducing them into heterodimers. Thus, these "wild-type bispecific molecules" represent a mixture of AA, AB and BB. All bispecific molecules in this study retain the same heavy and light chain combinations, ensuring that any observed effects are due to mutations in the Fc portion of the molecules and not due to changes in the Fab portion.

[0279] Hypothetically, the pairs of mutations used to promote heterodimeric pairing of two different heavy chains may be associated with unanticipated structural or other destabilizing effects on the Fc region of IgG, which may subsequently result in undesirable problems such as reduced in vivo half-life, reduced effector functions and / or increased immunogenicity due to the presence of these mutations that may hinder further clinical development.

[0280] (thermal stability) Stresses such as increasing or decreasing temperature can lead to conformational changes (e.g. unfolding) and / or aggregation of the protein. To investigate the thermal stability of CH3-engineered IgGs, combinations 3-6 and 9-12 (Table 15) were incubated with wild-type bispecific molecules and bispecific molecules obtained using constructs 1 and 2 (E356K:D399K / K392D':K409D', also called "charge-reversed" pairs) at 4, 60, 62.5, 65, 67.5, 70 and 72.5 °C for 1 h at 100 μg / ml (PBS in the solvent) in a PCR machine. After this, the samples were slowly cooled to 25 °C over 15 min and kept at this temperature for 2 h before being stored at 4 °C until the next day. The precipitated antibodies were removed by centrifugation (18,000 rpm; 4° C., 20 min), and the total IgG concentration of water-soluble antibodies was determined by Octet using a Protein A biosensor (1 / 10 diluted in PBS).

[0281] The results are shown in Figure 20. The control CH3 engineered bispecific antibody (charge-reversed combination E356K:D399K / K392D':K409D' (triangles)) has decreased thermal stability compared to the wild-type bispecific molecule (squares). Bispecific molecules from combinations 3-6 and 9-12 (diamonds) also demonstrated decreased thermal stability compared to the wild-type. Notably, however, three combinations demonstrated improved stability compared to the control CH3 engineered bispecific antibody. Bispecific molecules from combinations 9, 10 and 11 were significantly more stable than the other CH3 engineered (charge-reversed) bispecific molecules and were as stable as the wild-type bispecific molecule at the highest temperature measurement point.

[0282] (Freeze-thaw stability) To investigate the stability of CH3 engineered IgGs during repeated freeze-thaw cycles, bispecific molecules from combinations 3-6 and 9-12 (Table 15), wild-type bispecific molecules and bispecific molecules obtained using constructs 1 and 2 (E356K:D399K / K392D':K409D' combinations (charge-reversed pairs)) were subjected to 10 freeze-thaw cycles. For each cycle, samples were placed at -80°C for at least 15 min until completely frozen and then thawed at room temperature. Once completely thawed, the freeze-thaw cycle was repeated again. After 10 freeze-thaw cycles, precipitated antibodies were removed by centrifugation (18000 rpm; 4°C, 20 min) and the total IgG concentration of water-soluble antibodies was derived by Octet using a Protein A biosensor (1 / 10 PBS dilution). The freeze-thaw stability test was repeated three times.

[0283] The results are shown in Figure 21. The control charge-reversed CH3 engineered bispecific antibody appeared to have slightly decreased stability compared to the wild-type bispecific molecule. In contrast, the bispecific molecules from combinations 3, 4 and 9 appeared to have slightly improved stability compared to the wild-type bispecific molecule. Thus, it was concluded that the harsh conditions of freeze-thaw cycling do not pose significant stability issues for the CH3 engineered variants.

[0284] In vitro serum stability To investigate the stability of CH3 engineered IgG in serum maintained at 37°C, bispecific molecules from combinations 3-6 and 9-12 (Table 15) were incubated with wild-type and charge-reversed bispecific molecules in 10% human serum at 37°C. Control samples were kept at 4°C. After 1, 2, or 5 days, precipitated antibodies were removed by centrifugation. Samples were then spiked in a fibrinogen-specific ELISA to derive the relative amount of functional IgG. Purified control antibodies were acutely dosed in human serum and used as a reference group.

[0285] Based on the fibrinogen ELISA data, all samples were very stable after 5 days at 37° C. in 10% human serum. The bispecific molecules with lower IgG concentrations from combinations 4 and 5 were slightly less stable, especially at T=1 and T=2, but the differences were minimal at the end of the experiment ( FIG. 22 ).

[0286] Example 20: Further stability tests A series of further analytical methods were used to evaluate the stability of the mutant IgGs. Bispecific molecules from combinations 3-6 and 9-12 (Table 15), wild-type bispecific molecules (AA, AB, BB), the individual parental antibodies (AA and BB) and bispecific molecules obtained with constructs 1 and 2 (E356K:D399K / K392D':K409D' combinations (charge-reversed pair)) were used as samples for these stability assays.

[0287] All IgGs were diluted to 0.2 mg / ml and subjected to several stress conditions (50 °C for 2 days, 40 °C for 2 weeks, freeze-thaw cycles 5 times) to allow discrimination between different samples. Of note, these high stress levels lead to instability of one of the parent antibodies used in all bispecific molecules (the BB parent, carrying two 1122 Fabs). At 50 °C for 2 days, aggregation of this protein was detected by UV absorption, suggesting that this stress condition may not be able to distinguish between the instabilities of Fab and CH3 in the bispecific molecules. Therefore, data from the 50 °C incubation should be treated with caution.

[0288] The results are summarized in Table 16. The analytical methods used included: - fluorescence microscopy with Nile Red ("Nile Red Particles" in Table 16), to observe the amount of particles larger than 0.5 μm after adding Nile Red dye; - UV spectroscopy at 350 nm ("UV350nm"), changes in absorption at wavelengths longer than 320 nm provide information about the aggregation state of the protein; - 90° light scattering at 400 nm ("LS400nm"), a sensitive technique to observe changes in protein aggregation, e.g. the difference between monomeric and dimeric IgG; - autofluorescence, the fluorescence wavelength maximum and intensity of aromatic residues of proteins change depending on the environment (e.g. unfolding); - 1,8-ANS fluorescence spectroscopy, 1,8-ANS binds to cationic groups through electrostatic interactions via ion pair formation. Changes in protein structure and / or conformation are detected.

[0289] (UV-Vis spectroscopy) UV-Vis absorption spectra were measured on a Cary 300 Bio spectrophotometer with a double beam and two monochromators using different Varian quartz cuvettes (e.g., a black low-volume Hellma cuvette with 1.0 cm path length and a 0.2 cm x 1.0 cm clear Hellma cuvette) at 25°C. A 1.0 cm path length was used to monitor the spectrum between 220 and 450 nm. The absorption around 280 nm provides information on the protein concentration. The region between 320 nm and 450 nm provides information on the aggregation state of the sample.

[0290] (90° light scattering) A 90° light scattering spectroscopy method was developed to study protein aggregation. The method was performed similarly to that described in Capelle 2005 and Demeule 2007a. 90° light scattering spectra were monitored at 25°C using a FluoroMax fluorometer (Spex, Instruments SA, Inc. UK) running a synchronous scan (λem = λex) from 400 nm to 750 nm with an integration time of 0.01 s. Different slit settings were tried to obtain optimal conditions. Once optimized, all measurements were performed using that slit setting.

[0291] (Steady-state fluorescence emission) The fluorescence emission of tryptophan, tyrosine, and phenylalanine residues gives a picture of the local environment of these fluorophores. The change or difference in hydrophilicity and / or rigidity is measured. Generally, a more hydrophobic and rigid environment leads to an increase in the fluorescence intensity and a blue shift of the emission maximum. Autofluorescence spectroscopy provides information on the current state of the protein and monitors changes in physical and chemical properties. More detailed information on tyrosine and tryptophan can be found in Lakowicz's book (Lakowicz, 2006).

[0292] Fluorescence emission and excitation spectra were recorded at 25 °C in different quartz cuvettes. Samples were excited at different wavelengths. The integration time and slit settings were optimized. After optimization, the integration time and slit settings were applied to all samples.

[0293] Fluorescence microscopy with Nile Red staining. Nile Red staining was developed to visualize protein aggregation. The staining was performed as described by Demeule et al., 2007b.

[0294] Microscopic observations were performed with 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. The objectives were 10x, 20x and 40x. A fixed distance of 0.1 mm between the slide and the cover slip was used for the microscopic studies. The size of the 4x4 grid was 1mmx1mm, which corresponds to 0.1μl.

[0295] (1,8-ANS fluorescence spectroscopy) 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS) is an uncharged, hydrophobic, fluorescent small molecule (molecular weight 299.34 Da) that is used to study both membrane surfaces and proteins.

[0296] 1,8-ANS is virtually nonfluorescent in water and only fluoresces appreciably when bound to membranes (quantum yield ~0.25) or proteins (quantum yield ~0.7). This property makes 1,8-ANS a sensitive indicator of protein folding, conformational changes, and other processes that change upon exposure of the probe to water. Information about 1,8-ANS is available from Molecular Probes at www.probes.com.

[0297] The fluorescence emission spectra of 1,8-ANS were recorded using a FluoroMax fluorometer. Direct comparison of 1,8-ANS fluorescence between different IgGs was not performed because each IgG has a different number of binding sites for 1,8-ANS. In principle, the lower the 1,8-ANS fluorescence, the fewer 1,8-ANS molecules are bound to the antibody. Stress-induced changes in 1,8-ANS fluorescence intensity and emission wavelength were evaluated.

[0298] [Table 16]

[0299] Taken together, these data show that the various IgG samples are remarkably stable. Severe stress conditions (e.g., 50°C for 2 days) are required to produce measurable differences between the samples tested. Under these conditions, samples from combinations 9 and 10 appear to be more prone to aggregation than the other samples.

[0300] The most important factors that could differentiate the stability between proteins are freeze-thaw cycles and temperature elevation. Taking into account the very severe stress factor of incubation at 50°C, the two mutants T366K / L351E,Y349E (combination no. 4) and T366K,L351K / L351D,Y349E (combination no. 11) are the most stable proteins in this group, closely followed by T366K,L351K / L351D,Y349D (combination no. 10) and T366K,L351K / L351D,L368E (combination no. 12).

[0301] (Example 21: Experiments performing native MS at different ratios; transfection ratios from 1:5 to 5:1) To gain more knowledge about the behavior of CH3 mutated IgGs in mixtures when transfected at biased ratios, we performed more detailed ratio studies, especially for the T366K:L351K / L351D':L368E' combination (hereafter referred to as KK / DE or DEKK).

[0302] The VH regions of previously used antibodies with known specificity and binding ability to the known common light chain human IGKV1-39 were used to reclone into constructs 1, 2, 68 and 69, resulting in vector IV (Table 17). Vector IV, containing nucleic acid sequences encoding Ig heavy chains with different CH3 regions and different antigen specificities and the common human light chain, respectively, was then transfected into cells at different transfection ratios shown in Table 18. The results are shown in FIG. 23.

[0303] [Table 17]

[0304] [Table 18]

[0305] As shown in Figures 23A and B, in DEKK combination mutations, when an excess of A or C is present (A or C on the "DE side" and B on the "KK side"), AB or BC is formed, but in all cases the excess A or C exists as a mixture of both homodimers and half molecules. However, when an excess of B is present (B on the "KK side" and A or C on the "DE side"), a clear difference is seen: AB or BC are still formed, but the excess of B is not present as a homodimer at all, and only half molecules are formed.

[0306] It should be noted that the percentages are again measured by peak height. Peaks detected at or below 2% are below the threshold that the nMS technique used here can accurately measure. Therefore, measurements below 2% were considered to be within the noise level of the analysis and were ignored.

[0307] It is noteworthy that the excess of B results in a higher proportion of only the B half molecule. In particular, for A:B ratios of 1:3 and 1:5, a high proportion of B half molecules was observed without the presence of homodimer BB (Figures 23A and 23B), indicating that the mutations in CH3 on the KK side make homodimerization less likely. The absence of homodimerization is a crucial advantage, since when the "KK side" of the DEKK combination is chosen to add specificity, it is possible that known adverse effects may occur when present as homodimers (e.g., cMET or CD3 antibodies are known to have undesirable adverse side effects when present in pharmaceutical compositions as bivalent homodimers).

[0308] The observation of different ratios of DE:KK contrasts with the control charge-reversal CH3 mutations in vectors IV and V. As shown in Figure 23C, in the mutations of the E356K:D399K / K392D':K409D' combination, when A is present in excess (A is the "K392D:K409D side"), in all cases the excess A exists as a mixture of both homodimers and half molecules. When B is present in excess (B is the "E356K:D399K side"), in all cases the excess B exists as a mixture of both homodimers and half molecules. Even at higher ratios of 1:3 and 1:5, homodimers are present but half molecules B are not observed. This indicates that the E356K:D399K side does not favor homodimers as much as the KK side of the DEKK combination.

[0309] Taken together, the DEKK combination mutations have a distinct advantage over the charge-reversal CH3 mutations in that one of the chains of the heterodimer does not form a homodimer.

[0310] Example 22: Various mixtures using DEKK combinations Mutation of the DEKK combination was shown to induce the formation of bispecific IgG molecules ("AB") with high purity. We next explored the feasibility of controlled production of more complex antibody mixtures, such as "AB and AA" or "AB and AC" mixtures from a single cell. Previously used Fab types were incorporated into vectors containing either the "DE construct" or the "KK construct". Then, to demonstrate the versatility of the technology, various combinations of these vectors were co-expressed to generate mixtures. The Fab types MF1337 (tetanus toxin), MF1122 (fibrinogen) and MF1025 (thyroglobulin) were selected due to their overall stable behavior, good expression levels and mass differences between the IgGs containing these Fabs (see Table 19).

[0311] [Table 19]

[0312] [Table 20]

[0313] SDS-PAGE analysis showed that most samples consisted mostly of full-length IgG, with a small percentage of half-molecules in some cases. Furthermore, many samples showed two bands at approximately 150 kDa on gels under nonreducing conditions, reflecting the presence of two distinct IgG species in the samples. Two heavy chain bands were also visible in some samples on gels under reducing conditions (data not shown).

[0314] Native MS was performed on all samples and the percentage of observed species was calculated based on peak height (Percentage of "Observed Species" in Table 20). The results are shown in Figure 24. In all 8 samples where the three heavy chains were co-expressed, two major peaks corresponding to the expected species were observed. In two of these samples (transfections 2 and 4) and in transfection 11, small amounts of contaminating DE-DE homodimers were observed. Half molecules were detected in very low amounts in most samples (less than 2%). However, as mentioned above, this is not a problem as they can be easily separated from the full-length IgG portion.

[0315] After nMS, it was found that a different species was predicted for sample 11 than the one corresponding to the observed IgG mass, which was concluded to be a transfection error: it was apparent that 1337-KK, instead of 1122-KK, was co-expressed with 1025-DE in sample 11.

[0316] To confirm the functional presence of the desired specificity, IgG samples were further tested by sandwich ELISA. ELISA plates were coated with fibrinogen or thioglobulin, and detection was performed with fluorescein-labeled thioglobulin or tetanus toxoid. Detection antigens were labeled with fluorescein (Pierce NHS-fluorescin Antibody Labeling kit, cat. #53029) according to the manufacturer's instructions. Fluorescein-labeled antigens were then detected with FITC-conjugated anti-fluorescein antibody (Roche diagnostics, cat. # 11426346910).

[0317] A summary of the results of the dual specificity ELISA (OD450 values) is shown in Table 21. The grey cells indicate the expected species for each transfection. In general, the experimental results matched the expected results, with exceptions shown in italics or bold. For transfections 1-3, species BC (transfections #1 and #2) or AC (transfection #3) are putatively "negative" cells, but show significant background signal. From previous studies, it is known that dual specificity ELISAs suffer from high background levels. These background levels may be due to half molecules that may be present in the samples. Of note, the results of the dual specificity ELISA confirmed that an error had occurred in transfection #11, where species AC (bold values) was detected rather than BC.

[0318] [Table 21]

[0319] (Example 23: Improved mixture of two bispecific antibodies recognizing four different epitopes (AB and CD) from a single cell) In Example 12, there was a predicted hypothesis that the mixture from transfection ZA or ZB might be problematic when moving to mass production. The reason is that knob-into-hole mutants were reported to be unstable and it could not be excluded that CH3 domains with "knobs" or "holes" dimerize with charge-engineered CH3 domains. As shown in the above examples, novel charge pair mutants were found that selectively induce heterodimerization and virtually no homodimer formation. Polypeptide chains with CH3 domains with these novel charge pair mutants can be expressed in cells together with previously known polypeptide chains with charge-engineered CH3 domains or SEED bodies, resulting in the selective formation of only two bispecific molecules.

[0320] As is evident from the above examples, DEKK combination mutations are excellent for producing one bispecific molecule (AB) or two bispecific molecules (AB and AC) in clonal cells where heavy chain dimerization is driven by the CH3 domain. However, only one set of vectors can be used as complementary CH3 mutations, which limits the types of mixtures that can be produced. If there is a second "orthogonal" set of vectors that can be used in combination with DEKK, it is possible to produce more complex IgG and / or bispecific molecules such as "AB and CD" or "AB and CC" mixtures.

[0321] An important requirement when combining two sets of vectors is that the heavy chains expressed from two different sets of CH3 engineered vectors do not form "crossover" dimers, where heavy chains produced by one set of vectors dimerize with heavy chains expressed by the other set of vectors to form full-length IgG.

[0322] To test whether such "crossover" dimers can form, in silico analysis was performed using HADDOCK to gain insight into the possible pairing between the wild-type CH3 domain and the CH3 domain containing DE- or KK-mutations. Similarly, the possible pairing between the wild-type CH3 domain and the CH3 domain containing E356K, D399K or K392D, K409D mutations, between the wild-type CH3 domain and the CH3 domain containing knob-into-hole mutations, and any combination of the above were analyzed. The list of CH3 mutation combinations analyzed by HADDOCK is shown in Table 22. The resulting HADDOCK scores are summarized in Figure 25.

[0323] [Table 22]

[0324] Based on these HADDOCK predictions, it appears that the most successful combination is the CH3 of the DEKK combination with the CH3 of the charge-reversal combination, as shown in Figure 25. This combination allows the formation of two bispecific molecules of the desired combination (AB and CD) without contaminating by-products (especially AC, AD, BC, and BD) when co-expressed in a single cell.

[0325] As can be seen in Figure 25, these undesirable bispecific species AC, AD, BC and BD have relatively high HADDOCK scores, whereas the desirable AB and CD have the lowest HADDOCK scores. Of course, when either the DEKK or charge-reversed CH3 combinations are inserted into constructs that retain 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), CC and AB are predominantly produced when co-expressed in cells.

[0326] In contrast, when we look at the predictions for co-expression of DEKK and wild type, the HADDOCK scores for AC and AD are lower than the HADDOCK score for CD, indicating that AC and AD are highly likely to be contaminated when attempting to produce a mixture of AB and CD by co-expressing vectors encoding the DEKK CH3 combination together with a vector encoding the wild type CH3.

[0327] Finally, predictions for co-expression of either the DEKK or charge-reversal mutants together with knob-into-hole mutants resulted in undesirable dual-specific mutants having relatively low HADDOCK scores, indicating a high probability of producing these undesirable species upon co-expression.

[0328] It was therefore concluded that the combination of the CH3 of the DEKK combination with the CH3 of the charge reversal combination (E356K,D399K / K392'D,K409D') is ideally preferred for obtaining substantially pure mixtures of "AB and CD" and / or "AB and CC" antibodies.

[0329] Next, to put the above results into practice, a mixture of two bispecific molecules recognizing four targets / epitopes (AB and CD) and a mixture of one bispecific and one monospecific molecule recognizing three targets / epitopes (AB and CC) were generated. In generating these mixtures, all four different VHs can pair with the common light chain IGVK1-39, but each individual VH / VL combination has a different specificity.

[0330] To allow native MS analysis, the mass difference between the (predicted) species must be sufficient, i.e., greater than 190 Da. The four individual VHs were selected such that the mass values ​​of the predicted species upon co-expression would allow them to be identified and separated by nMS. Furthermore, the four selected VHs have a large enough mass difference to identify the two desired species as well as most of the contaminants that may occur in the mixture. The list of selected VHs is shown in Table 23.

[0331] [Table 23]

[0332] As shown in Table 24, four different VHs were cloned into vectors containing "DE" or "KK" constructs or charge-reversed constructs, and some were co-expressed. As before, all vectors also contain nucleic acid encoding the common light chain IGKV1-39. As shown before, when combining two sets of vectors, an important requirement is that the heavy chains expressed from two different sets of CH3 engineered vectors do not form "crossover" dimers. "Crossover" means that the heavy chains produced from one set of vectors dimerize with the heavy chains expressed by the other set of vectors to form full-length IgG. Control transfections were performed to test the possibility of "crossover" dimer formation between heavy chains containing charge-reversed mutations and heavy chains containing DE or KK mutations.

[0333] [Table 24]

[0334] Table 25 provides a further summary of the expected species masses and possible contaminants present in transfection numbers 9-11 of Table 24.

[0335] [Table 25]

[0336] All purified protein samples from transfections #1-11 were analyzed by SDS-PAGE, including three control samples (Figure 26). Additionally, MS analysis was performed on protein samples from transfections #9-11 to identify all species in the samples.

[0337] As can be seen in Figure 26, transfection #3 and transfection #4 resulted in the expected mismatch between the "KK" construct and either "E356K:D399K" or "K392D:K409D." The amount of half molecules in the protein samples from these transfections exceeded that of a full-length IgG molecule.

[0338] Transfections #7 and #8 resulted in approximately equal amounts of half molecules and full-length IgG in the protein samples. However, it is not possible to deduce from SDS-PAGE whether the full-length IgG represents the DE / DE dimer, the DE / E356K:D399K dimer, or the DE / K392D:K409D dimer. Notably, virtually no half molecules were observed in samples from transfections #9-11.

[0339] Figure 27 shows the results of nMS analysis of transfections #9 and #11. The percentage of expected species and contaminating species were calculated by peak height. In transfection #9, the expected species "AB and CD" represented 97% of the mixture (30% AB and 67% CD), while there is only about 3% of contaminating BD (Figure 27A). In transfection #11, the expected species "AB and CC" represented 94% of the mixture (33% AB and 61% CC), while there is only 6% of contaminating BC (4.1%) and AC (1.8%) (Figure 27B).

[0340] These data show that when a second set of "orthogonal" vectors is used in combination with DEKK, it becomes possible to produce more complex mixtures of IgG and / or bispecific molecules, such as "AB and CD" or "AB and CC" mixtures. When charge-reversal and DEKK constructs are used in combination together, only a very limited amount of "crossover" dimer formation occurs. It is expected that by adjusting the transfection ratio, these low percentage contaminating by-products can be further reduced.

[0341] Example 24: Single-dose pharmacokinetic study in mice To study the pharmacokinetic (pK) behavior of bispecific antibodies carrying a combination of DEKK mutations in their CH3 regions, this study measured and compared the pK parameters of three different IgG batches, including: 1) wild-type anti-tetanus toxin parent antibody 1337:1337 (two MF1337 Fabs in a wild-type Fc backbone), 2) wild-type anti-tetanus toxin parent antibody 1516:1516 (two MF1516 Fabs in a wild-type Fc backbone), and 3) a CH3-engineered bispecific anti-tetanus toxin antibody 1516:1337 carrying a combination of DEKK mutations in the Fc region (MF1516 Fab on the DE side and MF1337 Fab on the KK side).

[0342] The DEKK-bispecific antibody products were selected so that the parent antibodies had specificities of 1337:1337 and 1516:1516, because based on previous studies, there was no known pre-formulation serological response to these antibodies in some mouse strains, which would have made the results difficult to interpret. In addition, there was sufficient mass difference between the parent antibodies to allow for the discrimination of 1337:1337 (wild type Fc), 1516:1337 (DEKK Fc), and 1516:1516 (wild type Fc) by nMS.

[0343] Three IgG batches were prepared as previously described, except that the DNA used for transfection was made using an endotoxin-free maxiprep kit to ensure that the amount of endotoxin was as low as possible. The batches were then tested for protein concentration, aggregation, endotoxin content, and percentage of bispecific product. They were shown to meet the acceptance criteria for use of the IgG batches in subsequent pK studies: IgG concentration after gel filtration was greater than 0.3 mg / ml, aggregation was less than 5%, endotoxin content was 3 EU / mg protein, and the DEKK batch contained greater than 90% bispecific IgG.

[0344] Native mass spectrometry of the samples after gel filtration showed a high proportion of the expected species. A small amount of DE:DE homodimer estimated at approximately 2% was detected in sample 1516:1337 (Figure 28). It was concluded that the three IgG batches met the criteria for use in pK studies.

[0345] For comparison of pK parameters between the three batches, three groups of female C57BL / 6J mice (Harlan, The Netherlands) were administered 1 mg / kg human IgG (5 ml / kg immunoglobulin solution / kg body weight). The animals were 7-8 weeks old at the time of administration and weighed approximately 18-20 grams. Blood samples were collected pre-administration, 15 and 60 minutes after administration, and 2, 4, 8, 24, 48, 96, 168, 268, and 336 hours after administration. Serum samples were prepared and stored at temperatures below -20 degrees until analysis. Each group consisted of three subgroups of four mice, i.e. 12 mice / group. Samples were collected from each mouse at six time points.

[0346] Mice welfare was maintained in accordance with Directive 86 / 609 / EEC (the general principles governing the use of animals in experiments of the European Communities) and Dutch legislation (The Experiments on Animals Act, 1997). The study also complied with the Standards for Humane Care and Use of Laboratory Animals issued by the Division of Laboratory Animal Welfare of the National Institutes of Health, USA, identification number 45859-01 (expiry date: 30 April 2015).

[0347] Mice in group 1 received monospecific IgG antibody 1516:1516 full length (triangles). Mice in group 2 received monospecific IgG antibody 1337:1337 full length (squares). Mice in group 3 received bispecific IgG antibody 1516:1337 full length with a DEKK engineered CH3 region (1516 on the DE side and 1337 on the KK side) (diamonds).

[0348] Quantitative analysis of monoclonal human antibodies in mouse serum was performed by ELISA assay using Quantitative Human IgG ELISA (ZeptoMetrix, NY USA; ELISA kit No. 0801182). Briefly, the ELISA assay is based on the following principle: 96-well ELISA plates are coated with anti-human IgG, and human monoclonal antibodies bind to the anti-human IgG. The bound antibodies were then visualized using horseradish peroxidase (HRP)-conjugated polyclonal anti-human IgG antibodies.

[0349] The optical density (OD) of each well is directly proportional to the amount of antibody in the serum sample. The results are shown in Figure 29. It was observed that the full-length bispecific IgG carrying the combination of DEKK mutations and the parental monospecific antibodies are remarkably similar. The CH3 mutations present in the DEKK-bispecific antibody do not alter the stability or half-life, and the DEKK mutants behave like wild-type IgG.

[0350] (References) TIFF2024180421000028.tif177167

Claims

1. 1. A method for producing at least two different Ig-like molecules from a single host cell, comprising: each of said two Ig-like molecules comprises two CH3 domains capable of forming an interface; The method further comprises administering to the cell: a. a first nucleic acid molecule encoding a polypeptide chain comprising a first CH3 domain; b. a second nucleic acid molecule encoding a polypeptide chain comprising a second CH3 domain; c. a third nucleic acid molecule encoding a polypeptide chain comprising a third CH3 domain, and d. a fourth nucleic acid molecule encoding a polypeptide chain comprising a fourth CH3 domain; including providing providing means for selective pairing of a polypeptide comprising said first and second CH3 domains with a polypeptide comprising said third and fourth CH3 domains in at least two of said nucleic acid molecules; The method comprises: The method further comprises the steps of culturing said host cells to express said at least four nucleic acid molecules and recovering said at least two distinct Ig-like molecules from the culture.

2. 10. The method of claim 1 , The method further comprising providing said host cell with a nucleic acid molecule encoding a common light chain.

3. The method according to claim 1 or 2, the polypeptide chain comprising the first CH3 domain comprises the amino acid substitution T366K; The method of claim 1, wherein the polypeptide chain comprising the second CH3 domain comprises the amino acid substitution L351D.

4. 4. The method of claim 3, The polypeptide chain comprising the first CH3 domain further comprises the amino acid substitution L351K.

5. The method according to claim 3 or 4, The polypeptide chain comprising the second CH3 domain further comprises an amino acid substitution selected from the group of Y349E, Y349D and L368E.

6. 6. The method of claim 5, The polypeptide chain comprising the second CH3 domain further comprises the amino acid substitution L368E.

7. The method according to any one of claims 1 to 6, the polypeptide chain comprising the third CH3 domain comprises the amino acid substitutions E356K and D399K; The method of claim 1, wherein the polypeptide chain comprising the fourth CH3 domain comprises the amino acid substitutions K392D and K409D.

8. The method according to any one of claims 1 to 7, The method of claim 1, wherein each of the polypeptide chains comprising the CH3 domain further comprises a variable region that recognizes a target epitope.

9. 9. The method of claim 8, A method according to claim 1, wherein each of the four variable regions of a polypeptide chain comprising the four CH3 domains recognizes a different target epitope.

10. 9. The method of claim 8, the variable regions of the polypeptide chains comprising the first and second CH3 domains recognize different target epitopes; The method of claim 1, wherein the variable regions of the polypeptide chains comprising the third and fourth CH3 domains recognize the same target epitope.

11. 11. The method of claim 10, The method of claim 1, wherein the target epitope recognized by the variable region of a polypeptide chain comprising the third and fourth CH3 domains is identical to the target epitope recognized by the variable region of a polypeptide chain comprising the first or second CH3 domain.

12. 11. The method of claim 10, The method of claim 1, wherein the target epitope recognized by the variable region of a polypeptide chain comprising the third and fourth CH3 domains is different from the target epitope recognized by the variable region of a polypeptide chain comprising the first or second CH3 domain.

13. 9. The method of claim 8, the variable regions of the polypeptide chains comprising the first and second CH3 domains recognize the same target epitope; wherein the variable region of the polypeptide chain comprising the third and fourth CH3 domains recognizes a second target epitope that is distinct from the target epitope recognized by the first and second variable regions.

14. 14. The method according to any one of claims 8 to 13, The target epitopes are located on the same target molecule.

15. 15. The method of claim 14, The method, wherein the target molecule is a water-soluble molecule.

16. 15. The method of claim 14, The method, wherein the target molecule is a membrane-associated molecule.

17. 14. The method according to any one of claims 8 to 13, The target epitopes are located on different target molecules.

18. 18. The method of claim 17, The method, wherein said different target molecules are expressed in the same cell.

19. 18. The method of claim 17, The method, wherein the different target molecules are expressed in different cells.

20. 18. The method of claim 17, The method, wherein the different target molecules are water-soluble molecules.

21. 18. The method of claim 17, A method wherein one target molecule is a water-soluble molecule and the second target molecule is a membrane-bound molecule.

22. 22. The method according to any one of claims 1 to 21, The method, wherein said at least two different Ig-like molecules are antibodies.

23. 10. The method of claim 1 , The method, wherein the selective pairing means comprises engineered complementary knob-into-hole mutations, disulfide bridges, charge mutations, or combinations thereof.

24. 24. The method of claim 23, The method of claim 1, wherein said selective pairing means is selected from Table B.

25. 10. The method of claim 1 , comprising means for selective pairing of a polypeptide comprising said first and second CH3 domains with a polypeptide comprising said third and fourth CH3 domains in all four of said nucleic acid molecules; The method of claim 1, wherein said means for selective pairing of polypeptides comprising said first and second CH3 domains is different from said means for selective pairing of polypeptides comprising said third and fourth CH3 domains.

26. 22. The method according to any one of claims 8 to 13, 17 to 19 or 21, The method, wherein at least one of said target epitopes is located in a tumor cell.

27. 22. The method according to any one of claims 8 to 13, 17 to 19 or 21, The method, wherein at least one of said target epitopes is located on an effector cell.

28. 28. The method of claim 27, The method, wherein the effector cell is a NK cell, a T cell, a B cell, a monocyte, a macrophage, a dendritic cell, or a neutrophil granulocyte.

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

30. 30. A mixture of at least two different Ig-like molecules obtainable by the method according to any one of claims 1 to 29.

31. 31. The mixture according to claim 30, A mixture, wherein the at least two different Ig-like molecules bind to different epitopes on the same antigen and / or different epitopes on different antigens.

32. 32. The mixture according to claim 30 or 31, The mixture, wherein the at least two different Ig-like molecules include at least one heterodimeric Ig-like molecule.

33. 33. The mixture according to any one of claims 30 to 32, A mixture, wherein two of said at least two different Ig-like molecules are heterodimeric Ig-like molecules.

34. 1. A recombinant host cell comprising a nucleic acid sequence encoding a polypeptide chain comprising at least a first, second, third, and fourth CH3 domain, A recombinant host cell comprising means for selective pairing of a polypeptide comprising said first and second CH3 domains with a polypeptide comprising said third and fourth CH3 domains, in at least two of said nucleic acid sequences.

35. 35. The recombinant host cell of claim 34, A recombinant host cell, wherein the host cell further comprises a nucleic acid sequence encoding a common light chain.

36. 34. A pharmaceutical composition comprising at least two different Ig-like molecules of any one of claims 30 to 33 and a pharma- ceutically acceptable carrier.

37. 37. The pharmaceutical composition of claim 36, A pharmaceutical composition, wherein the at least two different Ig-like molecules are produced by a recombinant host cell according to claim 34 or 35.

38. 1. A method of generating a host cell for producing at least two different Ig-like molecules, comprising: The method comprises: introducing into the host cell a nucleic acid sequence encoding a polypeptide chain comprising at least a first, second, third, and fourth CH3 domain; providing means for selective pairing of a polypeptide comprising said first and second CH3 domains with a polypeptide comprising said third and fourth CH3 domains, in at least two of said nucleic acid sequences; The method wherein said nucleic acid sequences are introduced sequentially or simultaneously.

39. 39. The method of claim 38, The method further comprising introducing into said host cell a nucleic acid sequence encoding a common light chain.

40. 40. A culture of a recombinant host cell according to claim 34 or 35, or of a recombinant host cell obtainable by the method of claim 38 or 39, which produces at least two different Ig-like molecules.

Images