Methods and means for production of ig-like molecules

Abstract

To provide methods and means for improved and / or alternative technologies for producing biological therapeutics in the form of mixtures or bispecific approaches for targeting multiple disease-modifying molecules.SOLUTION: A method for producing a heterodimeric Ig-like molecule from a single host cell is provided, wherein the Ig-like molecule comprises two CH3 domains that are capable of forming an interface, the method comprising providing in the cell a. a first nucleic acid molecule encoding a first CH3 domain-comprising polypeptide chain, b. a second nucleic acid molecule encoding a second CH3 domain-comprising polypeptide chain, the first CH3 domain-comprising polypeptide chain comprising at least one substitution of a neutral amino acid residue with a positively charged amino acid residue, the second CH3 domain-comprising polypeptide chain comprising at least one substitution of a neutral amino acid residue with a negatively charged amino acid residue, the method further comprising culturing the host cell, expressing the two nucleic acid molecules, and harvesting the heterodimeric Ig-like molecule from the culture.SELECTED DRAWING: None

Description

Technical Field

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

Background Art

[0002] Currently, many widely used biological agents are isolated human or humanized recombinant monoclonal antibodies. These agents enhance the ability of the body's immune system to disable or remove cells and / or molecules involved in the disease process, or to eradicate invading pathogens or infected agents.

[0003] Monoclonal antibodies bind to a specific site of an antigen, i.e., an epitope. In therapeutic antibodies, the epitope is selected to perform desirable functions such as removing tumor cells, inhibiting receptor-ligand interactions, or neutralizing viruses.

[0004] Currently, approximately 30 monoclonal antibodies have been approved by the FDA. These antibodies are usually produced in large quantities, and their biophysical and biochemical properties are examined in detail, so that the quality of each batch is maintained and meets the standards.

[0005] Despite these advantages, monoclonal antibodies have some disadvantages. Some of them are related to the single specificity, which is an inherent feature of monoclonal antibodies, and the complexity of diseases.

[0006] In the course of a disease, it is not uncommon for many factors to be involved. Moreover, the actions of each factor related to the disease can be redundant or synergistic, and the expression of another receptor may increase, leading to crosstalk between signal transduction networks. Therefore, inhibiting multiple different elements and reaction pathways related to the disease may lead to an improvement in the efficiency of treatment. Due to the single specificity inherent in monoclonal antibodies, monoclonal antibodies can only interfere with one step in the complex disease process and may not achieve sufficient effects.

[0007] Apart from the argument of the multifaceted nature of the disease process, it has become clear that targeting only a single cell, a water-soluble protein, or a single epitope of a pathogen is not sufficient to achieve a satisfactory therapeutic effect for the disease. The reason is that the monoclonal antibody may no longer be able to bind to the target epitope and exert the desired effect. For example, tumor cells often evade treatment with monoclonal antibodies by reducing the expression of the target epitope on the growth factor receptor, introducing mutations, or shielding it.

[0008] By activating other receptors and / or their ligands, tumor cells activate alternative reaction pathways to continue growing and metastasizing. Similarly, viruses and other pathogens also evade treatment with monoclonal antibodies by introducing mutations, deletions, or shielding of the target epitope.

[0009] Monoclonal antibodies that bind to a single epitope do not generate all of the effector mechanisms induced by polyclonal antibodies. Prominent examples of effector mechanisms here include; opsonization (making the antigen more susceptible to phagocytosis), steric hindrance (the antigen enclosed by the antibody is inhibited from reaching the host cell or mucosal surface), detoxification of toxins, aggregation, or precipitation (antibodies bound to some water-soluble antigens aggregate and are then removed), complement activation, and antibody-dependent cytotoxicity (the antibody causes the target cell to be killed by NK cells and neutrophils).

[0010] Polyclonal antibodies applicable to treatment can be obtained from pooled human sera. Such serum-derived therapeutic polyclonal antibodies are used for the treatment and prevention of viral infections such as rabies virus, cytomegalovirus, and RS virus, the inactivation of toxins such as tetanus toxin and botulinum toxin, or the prevention of anti-D immunization.

[0011] The wider use of serum-derived polyclonal antibody preparations is hampered by the fact that the plasma used as raw material can only be applied to a limited range of targets such as infectious diseases and toxins. Furthermore, the product depends on the blood supply situation by donors in terms of both quantity and compatibility, resulting in significant variation from batch to batch. In addition, screening techniques have not kept up with the ever-evolving viruses, and immunoglobulin preparations are accompanied by the risk of transmitting infectious diseases. Finally, due to the long processes such as blood collection, screening, and immunoglobulin purification, plasma-derived immunoglobulins are costly to produce.

[0012] A mixture of monoclonal antibodies can enhance the effectiveness of monoclonal antibodies while having no 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 (for example, a mixture of two monoclonal antibodies against the HER2 receptor, a mixture of two antibodies against the receptor EGFR, and two monoclonal antibodies against rabies virus).

[0013] In this field, combinations of two monoclonal antibodies have been shown to have additive or synergistic effects and to induce effector mechanisms not associated with either antibody alone. For example, mixtures of two monoclonal antibodies against EGFR or HER2 have been shown to kill tumor cells more potently. This is based on a combination of activities including promotion of cytotoxicity by immune system effectors, promotion of receptor internalization, and enhanced inhibition of signaling pathways downstream of the receptor.

[0014] In combination therapies based on two monoclonal antibodies, the component antibodies are produced separately and mixed at the protein level. The drawback of this method is the enormous cost of conducting clinical trials for each of the two antibodies and (partially) repeating the process for the combination. This can render combination therapies based on antibody combinations unacceptable in terms of cost.

[0015] Alternatively, two recombinant cell lines producing the component monoclonal antibodies can be mixed in a fermenter and the resulting antibody mixture purified as a single sample (WO 2004 / 061104). The drawback of this method is the difficulty in controlling the composition, and thus the poor reproducibility of the resulting recombinant polyclonal antibody preparations. In particular, it is difficult to account for the temporal changes in such compositions as the cells are cultured.

[0016] In the past decade, bispecific antibodies have evolved instead of using combinations of two antibodies. In the case of combinations of two antibodies, two different immunoglobulin molecules in the mixture bind to different epitopes, which may be on the same or different targets. In bispecific antibodies, this is achieved by a single immunoglobulin molecule.

[0017] By binding to two epitopes of the same or different targets, a bispecific antibody can exert the same effect as a combination of two antibodies that bind to the same epitope. Furthermore, bispecific antibodies in IgG format have two different monovalent binding sites combined in one molecule, while a mixture of two IgG antibodies has two different bivalent binding molecules combined in one sample, so different effects of these formats have also been observed.

[0018] From a technical and regulatory perspective, it is less laborious to develop a single bispecific antibody. The reason is that production, preclinical, and clinical trials only need to be carried out for a single molecule. Therefore, a therapy based on a single bispecific antibody is promoted by a less laborious and cost-effective drug discovery process and provides a more efficient antibody therapy.

[0019] Bispecific antibodies in IgG format, consisting of two heavy chains and two light chains, are produced in various ways. For example, bispecific antibodies can be produced by fusing two cell lines that secrete antibodies or by expressing two antibodies in one cell using recombinant DNA technology. According to these methods, multiple types of antibodies are produced. The reason is that the heavy chain corresponding to each antibody can form a monospecific dimer (also called a homodimer) containing a pair of two identical heavy chains with the same specificity, and a bispecific dimer (also called a heterodimer) containing a pair of two different heavy chains with different specificities.

[0020] Furthermore, the light and heavy chains from each antibody can pair randomly and result 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 expressed as bispecificity. However, even when using a common light chain, expressing two heavy chains and one common light chain in a single cell will result in three different antibody types. That is, two single-specificity '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 the mixture of parental antibodies and bispecific antibodies, and to reduce mispaired heavy and light chains. However, there is a need for a bispecific antibody format that eliminates or minimizes some of the above problems.

SUMMARY OF THE INVENTION

PROBLEMS TO BE SOLVED BY THE INVENTION

[0022] In summary, in the prior art, various techniques and methods have been provided for producing monoclonal antibodies, bispecific antibodies, mixtures of monoclonal antibodies, or mixtures of single-specificity and bispecific antibodies that can be applied to the treatment of patients.

[0023] However, as described above, these existing techniques and methods each have their own difficulties and limitations. Therefore, there is a need to create improved and / or different techniques for producing biological therapeutic agents in a mixture or bispecific approach that target multiple molecules for modifying diseases.

MEANS FOR SOLVING THE PROBLEMS

[0024] The present invention provides improved and / or alternative techniques, methods, and means for producing biological therapeutics by a mixture or bispecific approach targeting multiple disease-modifying molecules. The present invention further provides products and uses resulting from these methods and means.

[0025] In the prior art, various approaches have been described to facilitate the formation of a specific bispecific antibody of interest. These methods can reduce the composition of unwanted antibodies in the resulting mixture.

[0026] Regarding antibodies, the CH3-CH3 interaction is 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). Further, when two CH3 domains interact with each other, they are known to face each other at a protein-protein contact surface 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 is made via, for example, van der Waals forces, hydrogen bonds, water-mediated hydrogen bonds, salt bridges or other electrostatic forces, attractive interactions between aromatic side chains, disulfide bonds or other known forces.

[0028] Approximately one-third of the contacting amino acid side chains at the contact surface of the CH3 domain of human IgG1 have been shown to contribute to the majority of the folding and association of the domain. Other (adjacent) amino acid residues are also assumed to affect the interaction at this protein-protein contact surface.

[0029] In the prior art, approaches that interfere with the dimerization of the heavy chains of antibodies have been adopted. To promote heterodimerization rather than homodimerization, specific engineering of the CH3 domain has been applied. Examples of such manipulation of the CH3-CH3 contact surface are described, for example, in WO 1998 / 050431, Ridgeway et al., 1996, Merchant et al. 1998, and are 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 a protuberance at the contact surface of the first polypeptide and a corresponding cavity in the second polypeptide such that the protuberance is positioned within the cavity. This promotes the formation of heteromultimers and inhibits the formation of homomultimers.

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

[0032] Using only the knob-into-hole technology, the proportion of the bispecific antibody of interest in the mixture of two parental antibodies and the bispecific antibody can be increased to at most 87%. Merchant et al. successfully increased the proportion of the bispecific antibody in the mixture to 95% by introducing a disulfide bond added between two CH3 domains in the CH3-CH3 contact surface. Still, in order to use such a bispecific antibody as a drug, the bispecific antibody must be purified from the homodimer and made into a pharmaceutically acceptable diluent or excipient form. Purifying the heteromultimer from such a mixture has become a major problem because the physicochemical properties of the homodimer and the heterodimer are similar.

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

[0034] Another example of such an operation at the CH3-CH3 contact surface is provided by the heterodimeric Fc technology. This technology is involved in the design of bispecific and asymmetric fusion proteins by the invention of a strand-exchange engineered domain (SEED) CH3 heterodimer. These SEED-CH3 heterodimers are derivatives of human IgG and IgA composed of portions having alternating CH3 sequences of human IgA and IgG. As a result, a pair of complementary human SEED-CH3 heterodimers called SEED-bodies are obtained (Davis JH. et al., Protein Engineering, Design & Selection 2010(23)195-202; International Publication No. 2007 / 110205).

[0035] As yet another approach for producing the desired bispecific antibody, for example, as described in European Patent Application Publication No. 01870459, U.S. Patent Application Publication No. 2010 / 0015133, International Publication No. 2007 / 147901, International Publication 2010 / 129304, Gunasekaran et al (2010), and International Publication No. 2009 / 089004, it is based on the electrostatic manipulation of contact residues that originally have a charge at the CH3-CH3 contact surface.

[0036] In the mutations of the CH3 domain of the heavy chain described in these publications, the contact residues of the original charged amino acids are replaced with amino acid residues of the opposite charge (charge reversal strategy). This changes the polarity of the charges on the opposing contact surfaces of the Fc dimer, and when electrostatically compatible Fc chains are co-expressed, favorable attractive interactions can occur. Thus, the formation of the desired Fc heterodimer is promoted, while the formation of the undesired Fc homodimer is inhibited by repulsive charge interactions.

[0037] There are reports that four characteristic pairs of charged residues are involved in the interaction between the domains at the CH3-CH3 contact surface. These are D356 / K439’, E357 / K370’, K392 / D399’ and D399 / K409’ (the residues of the first chain and the residues of the second chain are separated by “ / ”, and the numbering follows that of the report by Kabat (1991) where the residue number of the second chain is represented by a prime symbol (‘)). Since the CH3-CH3 contact surface is two-fold symmetric, each characteristic pair of charged residues appears twice in the native IgG (i.e., the electrostatic interactions of K439 / D356’, K370 / E357’, D399 / K392’ and K409 / D399’ also exist at this contact surface).

[0038] Taking advantage of this two-fold symmetry, it has been shown that in the inversion of a single charge, such as K409D in the first chain or D399'K in the second chain, the formation of homodimers is reduced due to the repulsion of the same charge. By applying the inversion operation to different charges, this repulsive effect was further enhanced. It has been shown that the expression of different CH3 domains with inversions of different complementary charges induces heterodimerization, resulting in an increase in the proportion of bispecific species in the mixture.

[0039] By the approach described above, the proportion of bispecific antibodies produced from a single cell could be set to a value between about 76% and about 96%. One object of the present invention is to provide an even more improved proportion of the desired bispecific antibody in a method for producing a bispecific antibody from a single cell. According to the present invention, by using electrostatic manipulation techniques as one of the means, alone or in combination with other means, such as the knob-into-hole approach, the proportion of the further improved desired (bispecific) antibody can be achieved.

[0040] In one aspect, in a method for producing at least two different Ig-like molecules from a single host cell provided by the present invention, each of the two Ig-like molecules comprises two CH3 domains capable of forming a contact surface, and the method comprises introducing into 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, comprising providing, with at least two of said nucleic acid molecules, means for the selective pairing of a polypeptide comprising said first and second CH3 domains and a polypeptide comprising said third and fourth CH3 domains, the method further comprising culturing said host cell, expressing said at least four nucleic acid molecules, and recovering said 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 the disease process or pathogen invasion, replication, and / or spread.

[0042] The use of a mixture of multiple bispecific antibodies is particularly useful for the treatment of certain diseases. For example, during treatment with an antibody or small molecule drug, tumor cells use many different strategies to acquire resistance. Resistance can involve multiple cell surface receptors and water-soluble molecules, and it is thought to 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 therapeutic modality. Such a mixture of bispecific antibodies is preferably produced from a single cell to facilitate the drug discovery process. This makes the drug discovery process less laborious from a regulatory perspective, cost-effective from a pharmaceutical and clinical development perspective, and easily achievable.

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

[0045] One further object of the present invention is to provide a method and means for producing a defined mixture of bispecific antibodies from one cell. As will be described later, preferably, a method is provided that results in a mixture of (bispecific) antibodies of dimer IgG molecules at a ratio higher than at least 95%, at least 97% or 99%, regardless of the amount of monomeric by-products. Generally speaking, in cells where multiple native IgG molecules are produced, half-molecules (monomeric by-products) are present, but are easily removed by known size exclusion chromatography.

[0046] In one embodiment, in the method for producing a defined mixture comprising at least two different Ig-like molecules in a single cell, instead of one (bispecific) antibody of interest provided by the present invention, the formation of other dimer antibody species that are not desired is reduced or absent. The resulting mixture is clearly defined and its composition is controlled by the design of mutations in the CH3 domain. Furthermore, the regulation of the expression level and / or the different transfection ratios used for expression affect the composition of the mixture.

[0047] In the method according to the present 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 present invention provides a mixture of at least two different Ig-like molecules obtained by the method of the present invention.

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

[0049] Similarly, the meaning of "selective pairing of the polypeptides comprising the third and fourth CH3 domains" is that substantially all of the resulting dimers having a polypeptide comprising a third CH3 domain and / or a polypeptide comprising a fourth CH3 domain consist of dimers formed from a pair of a polypeptide comprising one third CH3 domain and a polypeptide comprising one fourth CH3 domain.

[0050] As a result, when a nucleic acid molecule encoding a polypeptide having four different (A, B, C, D) CH3 domains is introduced into a single cell, instead of a mixture of 10 different Ig-like dimers (AA, AB, AC, AD, BB, BC, BD, CC, CD, and DD), a mixture of mainly two specific Ig-like molecules is produced.

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

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

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

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

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

[0056] Notably, the ratio of the 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. It is possible to produce an optimized ratio of the antibody mixture using known means. For example, the expression level of the nucleic acid molecule, i.e., the ratio of the resulting produced Ig-like molecules, 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] Said means for selective pairing preferably consist of complementary knob-into-hole mutations, disulfide bridges, charge mutations including charge reversal mutations, or combinations thereof. Those skilled in the art will understand that said means for selective pairing can be selected from a specific range of types of mutations. That is, at least four nucleic acid molecules encoding polypeptide chains comprising CH3 domains are required as charge mutations as means for selective pairing.

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

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

[0060] In the method according to the invention provided by one aspect of the invention, said means for the selective pairing of a polypeptide comprising said first and second CH3 domains is different from said means for the selective pairing of a polypeptide comprising said third and fourth CH3 domains. The meaning of "different" here is that the means for the selective pairing of a polypeptide comprising the first and second CH3 domains is designed such that the selective pairing of the first and second strands is preferred. In this design, the interaction between the first and a polypeptide chain comprising the third and / or fourth CH3 domains is substantially prevented from occurring. In other words, the dimerization of a polypeptide comprising the first CH3 domain and said third or fourth polypeptide is in principle prevented or suppressed to an extent close thereto. The polypeptide comprising the third and fourth CH3 domains is either wild-type or comprises a means for selective pairing different from the means for the selective pairing of the first and second CH3 domains.

[0061] Recent research has focused, for example, on the production of a single bispecific antibody using knob-into-hole technology or mutations (inversions) of charged contact amino acids present in the CH3 domain. However, prior to the present invention, it has not been possible to produce a defined mixture of at least two (bispecific) Ig-like molecules without significantly co-producing other dimer by-products.

[0062] The present invention provides a method for efficiently controlling the production of a well-defined mixture of Ig-like molecules, which contains a high proportion of bispecific ones. In a desired system with two bispecific ones, a proportion of at least 95%, at least 97% or higher of the (two) bispecific ones is obtained. This means that at most 5%, at most 3% or less of monospecific bivalent by-products are obtained. It should be noted that the amount of monomeric by-products (i.e., half-molecules) is not very important. The reason is that these half-molecules can be easily separated by taking advantage of the difference in size.

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

[0064] The method further provided according to the present invention is such that the target epitopes recognized by the variable regions of the polypeptide chains comprising the third and fourth CH3 domains are the same, but different from the target epitopes recognized by the variable regions of the polypeptide chains comprising the first or second CH3 domain.

[0065] In another method, the variable regions of the polypeptide chains comprising the first and second CH3 domains recognize different target epitopes, and when 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 having specificity for AB and AA, or AB and BB, is created.

[0066] Provided by the method according to the invention is one in which 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 the culture of a single cell. Non-limiting examples of such clearly defined mixtures are mixtures of a bispecific antibody specific for AB and a monospecific antibody specific for AA. Another example is a mixture of a bispecific antibody specific for AB and a monospecific antibody specific for BB. Yet another example is a mixture of a bispecific antibody specific for AB and a monospecific antibody specific for CC. Again, suitable means and methods are provided for producing the desired mixture of antibodies, having at least 90%, preferably 95%, most preferably at least 97%, or exceeding 99% of the desired antibodies.

[0068] In another embodiment provided by 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 regions of the polypeptide chains comprising the third and fourth CH3 domains recognize a second target epitope different from the target epitope recognized by the first and second variable regions. This results in the production of monospecific Ig-like molecules having mainly 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 the two targets are too far apart to be bound by a single bispecific antibody. Producing a mixture of monospecific antibodies in a single cell can be advantageous because it can be considered a single therapeutic product.

[0070] In the art, the therapeutic effects and safety of various monospecific antibodies have already been proven and manufacturing approvals have been obtained. Production of a mixture of monospecific antibodies in a single cell can facilitate the testing of the effects and safety of some such mixtures, and reduce the effects and costs for regulatory approval and manufacturing. 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% has not yet been obtained. One of the other objects of the present invention is to provide a means and method for producing a well-defined mixture of homodimers that reduces the formation of bispecific antibodies to less than 5%.

[0071] The method according to the present invention is suitable for the production of the desired mixture of any bispecific and / or monospecific Ig-like molecules. Again, to produce a defined mixture containing more than two different Ig-like molecules, for example, nucleic acid molecules encoding polypeptides comprising the 5th and 6th (and 7th and 8th, etc.) CH3 domains can be further used.

[0072] Preferably, in the method according to the present invention, at least two CH3 domains are used, including at least one combination of mutations according to the present invention among 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 will be described in detail below.

[0073] In this book, the term "Ig-like molecule" means a proteinaceous molecule having at least one immunoglobulin (Ig) domain. The Ig-like molecule comprises a sequence having the function of at least one immunoglobulin CH3 domain, and 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 means for selective pairing and is used for the selective pairing of two proteinaceous molecules comprising the CH3 domain to design a desired heterodimeric binding molecule or a mixture of binding molecules. The binding moiety introduced into a proteinaceous molecule comprising a CH3 domain can be any binding means including those exemplified by 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 the variable region of an antibody (i.e., a combination of VH / VL). The variable region, which is part of a polypeptide chain comprising a CH3 domain, preferably shares a common light chain. In such a case, only the VH of the variable region differs, while the VL is substantially identical in all variable regions.

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

[0077] In a more preferred embodiment, the Ig-like molecule comprises the entire length of the Fc backbone. In the 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] As used herein, the term "antibody" refers to a proteinaceous molecule belonging to the class of immunoglobulins, which is a domain that binds to one or more epitopes on an antigen and includes molecules derived from the variable region of an antibody or having sequence homology. Known antibodies include several isotypes such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM. The antibodies according to the present invention may be of any isotype or functional derivative and / or fragments thereof. In a preferred embodiment, antibodies of the IgG isotype are produced as the Ig-like molecule. The reason is that IgG antibodies have a longer half-life compared to, for example, antibodies of other isotypes.

[0079] Antibodies produced by the method according to the present invention can have sequences from any origin, including murine and human sequences. The antibodies may be composed of sequences from one origin, such as all being derived from human antibodies, or may have sequences with two or more origins, such as resulting in so-called chimeric or humanized antibodies.

[0080] Therapeutic antibodies are preferably as close as possible to the antibodies originally possessed by the subject (for example, in the case of a human subject, human antibodies). The binding of an antibody is expressed from the viewpoints of specificity and affinity. Specificity determines which antigen or epitope the binding domain binds to. Affinity is a measure for evaluating the binding strength to a specific antigen or epitope. Specific binding is defined as binding with an affinity (K D ) of at least 1×10 -5 M, more preferably 1×10 -7 M, more preferably 1×10 -9 M or higher. Typically, for therapeutic monoclonal antibodies, 1×10-10 Those having an affinity for M or a higher affinity than that are used.

[0081] As used in this document, the term "antigen" means a substance or molecule that, when taken up in a living body, causes the production of antibodies by the immune system. Antigens can have various origins, including, in particular, pathogens, tumor cells or other abnormal cells, haptens, or self-tissues. At the molecular level, an antigen is characterized by its ability to bind to the antigen-binding site of an antibody. A mixture of antigens is also regarded as an "antigen". That is, according to those skilled in the art, a lysate of tumor cells or virus particles is often regarded as an "antigen", while a preparation of such tumor cell lysates or virus particles also exists as many antigenic determinants.

[0082] An antigen comprises at least one, and often two or more, epitopes. Here, the term "epitope" means a part of an antigen that is recognized by the immune system, specifically an antibody, B cell, or T cell. Epitopes are usually considered to be derived from non-self proteins, but sequences derived from the host can also be classified as epitopes.

[0083] The term "CH3 domain" is well known. The IgG structure has four chains consisting of 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 chain are called the Fc (Fragment crystallizable) portion, Fc fragment, Fc backbone, or simply Fc.

[0084] An IgG molecule is a heterotetramer having two heavy chains linked by disulfide bonds (-S-S-) at the hinge region and two light chains. The heavy chains dimerize through interactions at the contact surface of the CH3-CH3 domains and at the hinge region. The number of disulfide bonds in the hinge region 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. A part of its physiological function is the interaction with the complement system and specific receptors on the surface of various cells. The interaction between the CH3 domains of two individual heavy chains is known to play an important role in inducing dimerization of the heavy chains.

[0086] Therefore, the CH3 domain plays a leading role in the association of the heavy chains of an antibody. Also, the contact surface between CH3 domains contains more than 20 contact residues from each chain, which play a role in the CH3-CH3 interaction (Deisenhofer J., Biochemistry 1981(20)2361-2370; Miller S., J. Mol. Biol. 1990(216)965-973; Padlan, Advances in Protein Chemistry 1996(49)57-133).

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

[0088] The terms "contact residue", "contact amino acid", "contact surface residue", and "contact surface amino acid" as used herein generally refer to any amino acid residue present in the CH3 domain that may be related to the contact between domains. In this regard, in the calculation of the solvent accessible surface area (ASA) of the residues of the CH3 domain in the presence and absence of the second chain, there is a difference in ASA (>1 Å) in the calculations under the two conditions 2) can be calculated by known techniques, including the method of identifying residues shown as (Lee and Richards J. Mol. Biol. 1971(55)379) as contact residues. The residues identified as contact residues, according to the EU numbering system, 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, 439 (Table A).

[0089]

Table 1A

[0090] The contact residues of the CH3-CH3 contact surface may be charged amino acids or neutral amino acid residues. The terms "charged amino acid residue" or "charged residue" as used herein mean an amino acid residue having 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] The terms "neutral amino acid residue" or neutral residue as used herein refer 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, the “CH3-CH3 domain contact surface” or “CH3 contact surface”, “CH3-CH3 pairing”, “domain contact surface” or simply “contact surface” refers to the association as a result of the interaction of amino acid residues of two CH3 domains of different polypeptides each having a CH3 domain. That is, it is at least one interaction between the amino acids of the first CH3 domain and the amino acids of the second CH3 domain. Such interactions are, for example, van der Waals forces, hydrogen bonds, water-mediated hydrogen bonds, salt bridges or other electrostatic forces, attractive interactions between aromatic side chains, formation of disulfide bonds, or other known forces.

[0093] As used herein, said means for selective pairing of the polypeptide having the first and second CH3 domains and the polypeptide having the third and fourth CH3 domains may be any known means.

[0094] In one embodiment, at least one nucleic acid molecule encodes a CH3 domain in which large amino acid residues (i.e., “knobs” or “protrusions”), such as, for example, R, F, Y, W, I or L, are included at the positions of contact residues, while at least one other nucleic acid molecule encodes a CH3 domain in which small amino acid residues (i.e., “holes” or “cavities”), such as, for example, G, A, S, T or V, are included at the positions of complementary contact residues. The resulting CH3 domains are likely to pair with each other due to the three-dimensional conformations of the said contacting amino acids. Herein, the knob-into-hole technology has already been described in detail.

[0095] In yet another embodiment of the present invention, at least one nucleic acid molecule encodes a CH3 domain that contains an amino acid having an opposite charge compared to the wild type at the position of the contacting residue that was originally a charged residue, i.e., at the position that was originally K, H, R, D, or E, while at least one other nucleic acid molecule encodes a CH3 domain that contains an amino acid having an opposite charge compared to the wild type at the position of the complementary contacting residue that was originally a charged residue. The resulting engineered CH3 domains are prone to pair with each other due to the opposite charges of the contacting amino acids, while the same CH3 domains are less likely to pair due to electrostatic repulsion. It is difficult.

[0096] According to one embodiment, the mutations of CH3 described in European Patent Application Publication No. 01870459 (Patent Document 3), International Publication No. 2009 / 089004, Gunasekaran et al (2010) are used.

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

[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 encode novel mutations of the CH3 domain provided by the present invention. As detailed below, the present invention provides novel CH3 mutations that enable the production of a specific bispecific Ig-like molecule of interest without producing significant amounts of unwanted (dimeric) by-products. Furthermore, the present invention provides novel CH3 mutations that enable the production of a specific monospecific Ig-like molecule of interest without producing significant amounts of unwanted (dimeric) by-products. Accordingly, the use of at least one of these CH3 mutations according to the present invention is preferred.

[0100] As used herein, the terms "polypeptide", "polypeptide molecule" or "polypeptide chain" refer to a chain of amino acids covalently linked through peptide bonds. Proteins are 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. The polypeptides according to the present invention may undergo post-translational modification processes such as, for example, glycosylation. Accordingly, a polypeptide chain comprising the CH3 domain of the present invention refers to a polypeptide chain comprising at least the Ig·CH3 domain and may include those that have undergone post-translational modification.

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

[0102] Furthermore, the present invention provides a method for creating a host cell that produces at least two different Ig-like molecules, the method comprising introducing into the host cell a nucleic acid sequence encoding a polypeptide chain comprising at least first, second, third, and fourth CH3 domains, and comprising means for selective pairing of a polypeptide comprising the first and second CH3 domains and a polypeptide comprising the third and fourth CH3 domains with at least two of the nucleic acid sequences, wherein the nucleic acid sequences are introduced sequentially or simultaneously.

[0103] In a further aspect of the present invention, a method for creating a host cell for producing a heterodimeric Ig-like molecule, the method comprising introducing into the host cell a nucleic acid sequence encoding a polypeptide chain comprising at least first and second CH3 domains, wherein the polypeptide chain comprising the first CH3 domain comprises at least one substitution from a neutral amino acid residue to a positively charged amino acid residue, and the polypeptide chain comprising the second CH3 domain comprises at least one substitution from a neutral amino acid residue to a negatively charged amino acid residue, and the nucleic acid sequences are introduced sequentially or simultaneously. The method of creating 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, a recombinant host cell comprising a nucleic acid sequence encoding a polypeptide chain comprising at least first, second, third, and fourth CH3 domains, and comprising means for selective pairing of a polypeptide comprising the first and second CH3 domains and a polypeptide comprising the third and fourth CH3 domains with at least two of the nucleic acid molecules.

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

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

[0107] The "host cell" of the present invention may be any host cell capable of expressing a recombinant DNA molecule. For example, 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 mammalian 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 3A cells, hybridomas, tumor cells, immortalized primary cells, embryonic retinal cells such as W138, HepG2, HeLa, HEK293, HT1080 or PER.C6.

[0108] In the selection of an expression system, mammalian cell expression vectors and hosts are sometimes used so that the antibody is properly glycosylated. Human cell lines, preferably PER.C6, are advantageously used to obtain antibodies that match the glycosylation pattern in humans. The conditions for growing or proliferating cells (see Tissue Culture, Academic Press, Kruse and Paterson, editors (1973)) may be somewhat different from the conditions for expressing the recombinant product. Also, the process is usually optimized to increase the proportion of the product and / or the growth of the cells relative to each other by methods generally known to those skilled in the art.

[0109] General guidelines, procedures, and practical techniques for maximizing the productivity of mammalian cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach (M. Butler, ed., IRL Press, 1991). The expression of antibodies in recombinant host cells is widely described in the known literature (for example, European Patent Application Publication No. 0120694, European Patent Application Publication No. 0314161, European Patent Application Publication No. 0481790, European Patent Application Publication No. 0523949, U.S. Patent No. 4816567, International Publication No. 00 / 63403). Nucleic acid molecules encoding the light and heavy chains can be extrachromosomal copies and / or stably integrated into the host cell chromosome, with the latter being preferred.

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

[0111] When obtaining the expression of the sequence of a nucleic acid molecule encoding a polypeptide comprising a CH3 domain, it is known that a sequence capable of inducing such expression is functionally related to the sequence of the nucleic acid molecule encoding the polypeptide comprising a CH3 domain. By "functionally related" it is meant that the nucleic acid sequence encoding the polypeptide comprising a CH3 domain or its precursor can induce the expression of the polypeptide comprising a CH3 domain or its precursor, and is thus related to the sequence capable of inducing expression.

[0112] For example, useful expression vectors are already available, such as a series of products of Invitrogen's pcDNA vectors. When the sequence encoding the polypeptide of interest is appropriately inserted with respect to the sequences controlling the transcription and translation of the encoded polypeptide, the resulting expression cassette is useful for producing, in other words expressing, the polypeptide of interest.

[0113] Sequences for inducing expression include promoters, enhancers, etc., and combinations thereof. These need to be functional in the host cell, thereby inducing the expression of functionally related nucleic acid sequences. The promoter can be constitutive or regulatable, and can be obtained from various sources including viruses, prokaryotes or eukaryotes, or can be artificially designed.

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

[0115] Any promoter or enhancer / promoter capable of inducing expression of the sequence of interest in a host cell is suitable for the present invention. In one embodiment, the sequence capable of inducing expression comprises the region of the CMV promoter, preferably the nucleotide region from -735 to +95 of the CMV immediate early gene enhancer / promoter. As those skilled in the art will realize, the expression sequences used in the present invention will preferably be a combination of elements that stabilize or enhance expression, such as insulators, matrix attachment regions, STAR elements (International Publication No. 03 / 004704), etc. This can increase the stability and / or level of expression.

[0116] The production of proteins in recombinant host cells is widely described, for example, as set forth 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 consists of metabolizing, growing, dividing, and / or producing the protein of interest in those cells. This is accomplished by methods known to those skilled in the art and includes, but is not limited to, feeding the cells nutrients. Such methods include growing while attached to a surface, growing in suspension, or a combination thereof.

[0117] Some culture conditions can be optimized by known methods to optimize the protein production amount. Culturing is carried out, for example, in dishes, roller bottles, or reactors, by batch culture, fed-batch culture, continuous culture, culture by hollow fiber, etc. It is known that for large-scale (continuous) production of recombinant proteins by cell culture, it is preferable to grow the cells in suspension. It is also known that it is preferable to culture the cells under conditions without animal or human-derived serum or components of animal or human-derived serum. Thus, since there is no additional animal or human-derived protein from the medium, purification is facilitated and safety is enhanced. On the other hand, since synthetic media are optimal in terms of reproducibility, the system becomes very reliable.

[0118] 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. Such methods include immunoprecipitation, centrifugation, filtration, size exclusion chromatography, affinity chromatography, cation and / or anion exchange chromatography, hydrophobic interaction chromatography, and the like. Protein A or protein G affinity chromatography can be preferably used for a mixture of antibodies containing IgG molecules (see, for example, U.S. Patent No. 4,801,687 and U.S. Patent No. 5,151,504).

[0119] The Ig-like molecules and / or mixtures thereof produced by the method according to the present invention preferably have a common light chain. Accordingly, further provided is a method according to the present invention, further comprising the step of providing a nucleic acid molecule encoding a common light chain to the host cell. This is a light chain that can pair with at least two different heavy chains, thereby forming a functional antigen-binding domain. The functional antigen-binding domain can specifically bind 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 present invention. Thereby, in the formation of a functional antigen-binding domain, mispairing of incompatible heavy and light chains can be avoided. In one embodiment, a single common light chain of the same amino acid sequence can be used. In another way, those skilled in the art will recognize that functionally equivalent light chains can be included in the meaning of "common" even if the amino acid sequences are not identical. Many variants of the light chain exist, and these have mutations (deletions, substitutions, additions) but do not substantially affect the formation of the functional binding region. Thus, such variants can also bind to different heavy chains and form a functional antigen-binding domain.

[0121] As used herein, the term "common light chain" refers to a light chain that is either identical or has differences in amino acid sequence, but retains the binding specificity of the antibody formed as a result of pairing with a heavy chain. For example, by introducing and testing conservative amino acid changes and / or amino acid changes in regions that do not contribute or only partially contribute to the binding specificity when paired with a heavy chain, it is possible to create or find a functionally equivalent light chain even if it is not the same light chain.

[0122] The term "common light chain" includes a specific common light chain and combinations of 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 light chain derived from a germline-like source, more preferably a light chain derived from a germline, preferably a recombinant human germline-derived κ light chain, and most preferably a recombinant 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 of ordinary skill in the art may select means for forced pairing of heavy and light chains, such as those described in WO 2009 / 080251, WO 2009 / 080252, and / or WO 2009 / 080253.

[0124] The present invention provides not only novel combinations of engineered CH3 mutations but also novel engineered CH3 domains. Prior to the present invention, charged contact amino acids in CH3 domains known to be involved in CH3-CH3 pairing were substituted with amino acids of the opposite charge (charge reversal), thereby affecting CH3-CH3 pairing.

[0125] The mutations according to the present invention are a creation separate from this approach. The reason is that in wild-type CH3, uncharged or neutral CH3 amino acids are replaced by charged residues. In this embodiment of the present invention, instead of exchanging charged contact amino acids for amino acids of the opposite charge, uncharged CH3 amino acids are replaced by charged ones.

[0126] The approach of the present invention not only provides a way to efficiently promote dimerization of the CH3 domain, but also has the advantage that at least one additional charge-charge interaction is created at the CH3 contact surface. In addition to the pairs of charges present at the CH3-CH3 contact surface, this additional charge-charge interaction makes the dimers according to the present invention generally more stable compared to wild-type dimers (wild-type dimers are defined as bispecific IgG (AB) in which the CH3 has not been manipulated and are contrasted with parental homodimers (AA or BB)).

[0127] Also, surprisingly, it is possible to further increase the proportion of one or more of the desired Ig-like molecules in the mixture. As described above, generally, in known methods for preferentially producing bispecific antibodies, unwanted dimer by-products are produced. For example, when using the knob-into-hole technology, the proportion of the desired bispecific antibody is at most 87%. On the other hand, in the approach of electrostatic manipulation in which charged contact amino acids are replaced by amino acids of the opposite charge, the proportion is 96% (see, for example, Example 11).

[0128] Most surprisingly, the inventors of the present invention have succeeded in introducing mutations that further increase the proportion of the desired Ig-like molecules in the mixture. For example, in Example 17, it is shown that by the method using the mutations according to the present invention, a high proportion of the desired bispecific antibody is obtained such that no dimer by-products are detected at all in the resulting mixture. There are some unpaired half-molecules in which only one heavy chain is paired with a common light chain present in the mixture to some extent, but these are the result of unbalanced expression of the heavy chains and can be easily separated from the mixture by size exclusion chromatography.

[0129] Thus, due to such mutations according to the present invention, a high proportion of bispecific Ig-like molecules particularly suitable for pharmaceutical compositions are produced in single cells with substantially no contamination of dimer by-products.

[0130] In a method for producing a heterodimeric Ig-like molecule from a single cell, provided by a preferred embodiment of the present invention, the Ig-like molecule comprises two CH3 domains capable of forming a contact surface, and the method comprises introducing into 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 wherein the polypeptide chain comprising the first CH3 domain comprises at least one substitution from a neutral amino acid residue to a positively charged amino acid residue, the polypeptide chain comprising the second CH3 domain comprises at least one substitution from a neutral amino acid residue to a negatively charged amino acid residue, and the method further comprises 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 introducing into the host cell a nucleic acid molecule encoding a common light chain, and the general advantages thereof have been described above.

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

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

[0133] In the method further provided by the present invention, the means of selective pairing of the polypeptides comprising the first and second CH3 domains, or the means of selective pairing of the polypeptides comprising the third and fourth CH3 domains, is a substitution that makes threonine at position 366 of the first or third CH3 domain into a first charged amino acid and a substitution that makes leucine at position 351 of the second or fourth CH3 domain into a second charged amino acid, and the first and second charged amino acids have opposite charges.

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

[0135] As described above, the amino acids at position 366 of one CH3 domain and position 351 of the second CH3 domain have been reported to be a pair of contact residues at the CH3-CH3 contact surface. The lysine introduced at position 366 and the aspartic acid introduced at position 351 have opposite charges, and these amino acids electrostatically attract each other. Therefore, the first CH3 domain selectively attracts the second CH3 domain. Also, a pair of Ig-like molecules is predominantly formed between the first CH3 domain having lysine at position 366 and the second CH3 domain having aspartic acid at position 351.

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

[0137] As described above, one of the advantageous aspects of the mutations according to the present invention is that instead of replacing the interaction of the originally charged amino acids, a new interaction is generated between the newly introduced pair of charged amino acids. This aspect has not been disclosed or suggested heretofore.

[0138] In the method for producing at least two different Ig-like molecules according to the present invention from a single host cell provided by one aspect of the present invention, 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] In a method for producing a heterodimeric Ig-like molecule from a single cell provided by one embodiment, the Ig-like molecule comprises two CH3 domains capable of forming a contact surface, and the method comprises introducing into 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 comprising providing, wherein the polypeptide chain comprising the first CH3 domain comprises the amino acid substitution T366K, the polypeptide chain comprising the second CH3 domain comprises the amino acid substitution L351D, and the method further comprises culturing the host cell, expressing the nucleic acid molecule, and recovering the heterodimeric Ig-like molecule from the culture.

[0140] By using the above amino acid mutations according to the present invention, it becomes possible to produce a heterodimeric Ig-like molecule from a single cell. Thereby, the contamination of homodimers is less than 5%, preferably less than 2%, more preferably less than 1%, or most preferably the contamination of homodimers can be substantially eliminated.

[0141] In a method for producing a heterodimeric Ig-like molecule from a single cell provided by one embodiment, the Ig-like molecule comprises two CH3 domains capable of forming a contact surface, the presence of contaminating homodimers is less than 5%, preferably less than 2%, more preferably less than 1%, and most preferably there is substantially no contamination of homodimers. The method comprises 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 comprising providing, wherein the polypeptide chain comprising the first CH3 domain comprises the amino acid substitution T366K, the polypeptide chain comprising the second CH3 domain comprises the amino acid substitution L351D, and the method further comprises culturing the host cell, expressing the two nucleic acid molecules, and recovering the heterodimeric Ig-like molecule from the culture.

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

[0143] Accordingly, in a preferred embodiment, the above-described T366K / L351’D mutation according to the present invention can be further combined with a substitution that changes leucine (L) to glutamic acid (E) at position 368 of the second CH3 domain. This can be designated, for example, as the T366K / L351’D, L368’E mutation (however, other designations such as T336K / L351D-L368E or T366K / L351D, L368E or T366K-L351D,L368E are also possible).

[0144] As shown in Example 17, introduction of this mutation into a polypeptide comprising a first CH3 domain specific for antigen A and a polypeptide comprising a second CH3 domain specific for antigen B according to the present invention allows for obtaining bispecific Ig-like molecules with a two-sided AB specificity in particularly good yields. This pair of mutations results in the formation of undetectable amounts of homodimers and allows for obtaining bispecific antibodies.

[0145] In a method for producing a heterodimeric Ig-like molecule from a single cell provided by one preferred embodiment, the Ig-like molecule comprises two CH3 domains that can form a contact surface, the presence of contaminating homodimers is less than 5%, preferably less than 2%, more preferably less than 1%, and most preferably there is substantially no contaminating homodimer, and the method comprises introducing into 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 which comprises providing, wherein the polypeptide chain comprising the first CH3 domain comprises the amino acid substitution T366K, the polypeptide chain comprising the second CH3 domain comprises the amino acid substitutions L351D and L368E, and the method further comprises culturing the host cell, expressing the two nucleic acid molecules, and recovering the heterodimeric Ig-like molecule from the culture.

[0146] In another preferred embodiment, threonine (T) at position 366 of the first CH3 domain is replaced with lysine (K), leucine (L) at position 351 of the second CH3 domain is replaced with aspartic acid (D), and tyrosine (Y) at position 349 of the second CH3 domain is replaced with glutamic acid (E). This can be denoted, for example, as the T366K / L351’D,Y349’E mutation, or for example as T366K-L351D:Y349E or T366K / L351D,Y349E or simply T366K / L351DY349E.

[0147] The Y349 residue is a residue near the residue at position 351 that can contribute to dimer interaction. According to in silico data, Y349E leads not only to destabilization of the monomeric dimer (higher computational score) but also to a decrease in the stability of the heterodimer (lower computational score), and glutamic acid (E) is more preferred than aspartic acid (D) at position 349. Thus, introducing a second amino acid substitution into a polypeptide comprising a second CH3 domain that already has an amino acid substitution at position 351 facilitates heterodimerization.

[0148] In a method for producing a heterodimeric Ig-like molecule from a single cell, provided by a particularly preferred embodiment, the Ig-like molecule comprises two CH3 domains capable of forming a contact surface, the contamination of the homodimer is less than 5%, more preferably less than 2%, even more preferably less than 1%, and most preferably substantially absent, and the method comprises introducing into 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 comprising providing, wherein the polypeptide chain comprising the first CH3 domain comprises the amino acid substitution T366K, the polypeptide chain comprising the second CH3 domain comprises the amino acid substitutions L351D and Y349E, and the method further comprises culturing the host cell, expressing the two nucleic acid molecules, and recovering the heterodimeric Ig-like molecule from the culture.

[0149] In another preferred embodiment, threonine (T) at position 366 of the first CH3 domain is replaced with lysine (K), aspartic acid (D) at position 351 of the second CH3 domain is replaced with leucine (L), tyrosine (Y) at position 349 of the second CH3 domain is replaced with glutamic acid (E), and leucine (L) at position 368 of the second CH3 domain is replaced with glutamic acid (E). This is designated as the T366K / L351’D,Y349’E,L368’E mutation. The two residues Y349 and L368 are residues that may contribute to dimer interaction.

[0150] According to in silico data, Y349E and L368E not only lead to destabilization of the BB dimer (higher in silico score) but also to stabilization of the heterodimer (lower in silico score), and glutamic acid at positions 349 and 368 is more preferred than aspartic acid (D). Thus, by introducing second and third amino acid substitutions into the B-chain that already has an amino acid substitution at position 351, heterodimer formation is further promoted.

[0151] In a method for producing a heterodimeric Ig-like molecule from a single cell, provided by a particularly preferred embodiment, the Ig-like molecule comprises two CH3 domains that can form a contact surface, the contamination of homodimers is less than 5%, more preferably less than 2%, even more preferably less than 1%, and most preferably substantially absent, and the method comprises introducing into 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 comprises the amino acid substitution T366K, the polypeptide chain comprising the second CH3 domain comprises the amino acid substitutions L351D, Y349E and L368E, and the method further comprises culturing the host cell, expressing the two nucleic acid molecules, and recovering the heterodimeric Ig-like molecule from the culture.

[0152] In another preferred embodiment, threonine (T) at position 366 of the first CH3 domain is replaced with lysine (K), leucine (L) at position 351 of the first CH3 domain is replaced with lysine (K), leucine (L) at position 351 of the second CH3 domain is replaced with aspartic acid (D), and leucine (L) at position 368 of the second CH3 domain is replaced with glutamic acid (E). This is designated as the T366K, L351K / L351’D, L368’E mutation. As shown in the examples, this mutation also increases the proportion of the desired (bispecific) antibody. Furthermore, with this mutation, no detectable amount of homodimer formation occurs, and a bispecific antibody can be obtained.

[0153] Furthermore, in a method for producing a heterodimeric Ig-like molecule from a single cell, provided, the Ig-like molecule comprises two CH3 domains that can form a contact surface, the contamination of homodimers is less than 5%, more preferably less than 2%, even more preferably less than 1% and most preferably substantially absent, and the method comprises introducing into 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 comprising the step of providing, wherein the polypeptide chain comprising the first CH3 domain comprises amino acid substitutions T366K and L351K, the polypeptide chain comprising the second CH3 domain comprises amino acid substitutions L351D and L368E, and the method further comprises culturing the host cell, expressing the two nucleic acid molecules, and recovering the heterodimeric Ig-like molecule from the culture.

[0154] In another preferred embodiment, threonine (T) at position 366 of the first CH3 domain is replaced with lysine (K), leucine (L) at position 351 of the first CH3 domain is replaced with lysine (K), leucine (L) at position 351 of the second CH3 domain is replaced with aspartic acid (D), tyrosine (Y) at position 349 of the second CH3 domain is replaced with aspartic acid (D), and arginine (R) at position 355 of the second CH3 domain is replaced with aspartic acid (D). This is designated as the T366K, L351K / L351’D, Y349’D, R355’D mutation. This pair of T366K-L351K / L351’D-Y349’D is further improved by the R355’D mutation in the B-chain, increasing the in silico score for BB and slightly increasing the in silico score for AB.

[0155] Also provided, in a method for producing a heterodimeric Ig-like molecule from a single cell, the Ig-like molecule comprises two CH3 domains capable of forming a contact surface, the presence of homodimers is less than 5%, more preferably less than 2%, even more preferably less than 1%, and most preferably substantially absent, and the method comprises introducing into 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 comprising a step of providing, wherein the polypeptide chain comprising the first CH3 domain comprises amino acid substitutions T366K and L351K, the polypeptide chain comprising the second CH3 domain comprises amino acid substitutions L351D, Y349D and R355D, and the method further comprises culturing the host cell, expressing the two nucleic acid molecules, and recovering the heterodimeric Ig-like molecule from the culture.

[0156] Table B is a list of mutations introduced into the CH3 domain as a preferred means of selective pairing for the production of heterodimers or homodimers.

Table 1B

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

[0158] In the novel combination of CH3 mutations provided by the present invention, it is possible to produce a mixture of at least two monospecific Ig-like molecules in a single cell, with the contamination of bispecific Ig-like molecules being less than 5%, preferably more than 2%, more preferably less than 1%, and most preferably substantially absent. These mutations according to the present invention are particularly suitable for the production of mixtures of monospecific antibodies. This is when a high level of cross-linking of two identical target molecules is desired, when it is necessary to achieve a sufficiently high density of antibodies on the target cells to activate certain effector mechanisms such as complement-mediated lysis of tumor cells, or when the two targets are too far apart to be bound by a single bispecific antibody, or when it is particularly useful to simplify the procedures for 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 insights from the present invention, when lysine (K) at position 392 of a polypeptide having a first CH3 domain (e.g., having A specificity) is substituted with aspartic acid (D), aspartic acid (D) at position 399 of the polypeptide having the first CH3 domain is substituted with lysine (K), and lysine (K) at position 409 of the polypeptide having the first CH3 domain is substituted with aspartic acid (D), it is possible to produce in a single cell a mixture of at least two different monospecific Ig-like molecules including monospecific Ig-like molecules having AA specificity, and the formation of bispecific by-products (bispecific Ig-like molecules) is less than 5%, or less than 3%, or reduced to such an extent that it is substantially not detected at all.

[0160] Therefore, the above-mentioned combination of mutations (designated as K392D, D399K, K409D) is particularly preferred for the production of a mixture of single-specificity Ig-like molecules. Those skilled in the art will recognize that functional variants, namely K392E, D399R, K409E, can result in similar effects. In addition, double mutants comprising the substitutions of D399K and K409D, or K392D and K409D, D399R and K409E, etc., will also be able to exhibit similar effects.

[0161] The same is true for the combination of mutations when substituting glutamic acid (E) at position 356 of the polypeptide comprising the first CH3 domain with lysine (K), glutamic acid (E) at position 357 of the polypeptide comprising the first CH3 domain with lysine (K), lysine (K) at position 439 of the polypeptide comprising the first CH3 domain with aspartic acid (D), and lysine (K) at position 370 of the polypeptide comprising the first CH3 domain with aspartic acid (D). This combination of mutations (designated as E356K, E357K, K439D, K370D) is also particularly preferred for the production of a mixture of single-specificity Ig-like molecules.

[0162] Those skilled in the art will recognize that functional variants, namely K356R, E357R, K439E, K370E, can result in similar effects. In addition, triple or double mutants comprising the substitutions of E356K and K439D, E357K and K370D, or other functional variants will also be able to exhibit similar effects.

[0163] In a further embodiment, a method for producing at least two different single-specificity Ig-like molecules from a single host cell, each of the two Ig-like molecules comprising two CH3 domains capable of forming a contact surface, the method comprising introducing into 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 comprising the step of providing, wherein the polypeptide chain comprising the first CH3 domain comprises mutations of K392D, D399K and K409D, and the polypeptide chain comprising the second CH3 domain comprises a wild-type CH3 domain or comprises E356K, E357K, K439D and K370D mutations, and the method further comprises culturing the host cell, expressing the nucleic acid molecule, and recovering the at least two different Ig-like molecules from the culture.

[0164] In another embodiment, a method for producing at least two different single-specificity Ig-like molecules from a single host cell, each of the two Ig-like molecules comprises two CH3 domains capable of forming a contact surface, and the method comprises introducing into 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 comprising the step of providing, wherein the polypeptide chain comprising the first CH3 domain comprises a wild-type CH3 domain or comprises mutations of K392D, D399K, K409D, and the polypeptide chain comprising the second CH3 domain comprises E356K, E357K, K439D, K370D mutations, and the method further comprises culturing the host cell, expressing the nucleic acid molecule, and recovering the at least two different Ig-like molecules from the culture.

[0165] As shown in Example 10, two single-specificity Ig-like molecules are produced in a single cell and substantially no formation of bispecific Ig-like molecules is detected. One skilled in the art can select a third nucleic acid molecule encoding a polypeptide chain comprising a wild-type or engineered CH3 domain and provide it to the host cell such that a mixture of three single-specificity antibodies is produced.

[0166] In one aspect of the present invention, in the method for producing at least two different Ig-like molecules or the method for producing a heterodimeric Ig-like molecule according to the present invention, the polypeptide chains each having a CH3 domain further have variable regions that recognize different target epitopes, and the target epitopes are located on the same molecule.

[0167] Thereby, compared with the case where only one epitope is targeted, a more efficient antagonistic effect can be exerted on the (biological) function of the target molecule. For example, a heterodimeric Ig-like molecule can simultaneously bind to two epitopes present in a growth factor receptor or a water-soluble molecule important for tumor cell growth. Thereby, several independent signaling pathways can be efficiently inhibited, leading to uncontrolled growth. And in any combination of at least two Ig-like molecules, it can simultaneously bind to two, three, or four epitopes present in such a growth factor receptor or water-soluble molecule.

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

[0169] In another aspect of the present invention, in the method for producing at least two different Ig-like molecules or the method for producing a heterodimeric Ig-like molecule according to the present invention, the polypeptide chains each having a CH3 domain further have variable regions that recognize target epitopes, and the target epitopes are located on different molecules. In this case, each different target molecule can 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, and in other embodiments, different target molecules are expressed in different cells.

[0171] As a non-limiting example, 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 viral strains.

[0172] In the production method according to the invention of at least two Ig-like molecules or heterodimeric Ig-like molecules provided by one preferred embodiment, at least one of the target epitopes is located on a tumor cell. In another method, or in addition to this, at least one of the target epitopes is located on the surface of an effector cell. This is suitable for, for example, the recruitment of T cells or NK cells for the killing of tumor cells. For example, at least one Ig-like molecule produced by the method according to the invention can recruit immune effector cells, preferably human immune effector cells, by specifically binding to a target molecule located on the immune effector cells.

[0173] In a further embodiment, after the Ig-like molecule binds to the target molecule, the immune effector cells are activated. The induction of effector mechanisms includes, for example, the re-modulation of immune-regulated cytotoxicity by the Ig-like molecule produced by the method according to the invention. The Ig-like molecule can bind to a molecule that causes cytotoxicity, such as a T cell receptor or an Fcγ receptor, thereby activating downstream immune effector pathways.

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

[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 said target epitope is located on a CD3, CD16, CD25, CD28, CD64, CD89, NKG2D or NKp46 molecule. The viability of target cells includes the ability of the cells to survive, proliferate and / or interact with other cells.

[0176] In the method for producing a heterodimeric Ig-like molecule according to the invention, provided by one aspect of the invention, each polypeptide chain comprising a CH3 domain further comprises a variable region that recognizes 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 other embodiments, 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 molecule or a water-soluble molecule. In other embodiments, the different target epitopes are located on different target molecules, which are expressed on the same cell or 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 one 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., an NK cell, T cell, B cell, monocyte, macrophage, dendritic cell or neutrophil, and the target epitope is located on a CD3, CD16, CD25, CD28, CD64, CD89, NKG2D or NKp46 molecule).

[0179] In a preferred embodiment, in the method for producing at least two different Ig-like molecules or a heterodimeric Ig-like molecule according to the invention provided by the preferred embodiment, as described above, the at least two different Ig-like molecules are antibodies, most preferably antibodies of the IgG isotype, and even more preferably antibodies of the IgG1 isotype.

[0180] There is further provided an Ig-like molecule, a heterodimeric Ig-like molecule, or a mixture of at least two Ig-like molecules obtained by the method according to the invention. The (heterodimeric) Ig-like molecule or the mixture of Ig-like molecules preferably comprises at least one CH3 mutation described in Table B. There is provided not only a pharmaceutical composition comprising at least one Ig-like molecule or a mixture of at least two Ig-like molecules according to the invention, but also those comprising at least one mutation described in Table B in the (heterodimeric) Ig-like molecule or the mixture of at least two Ig-like molecules.

[0181] In one embodiment, the Ig-like molecule is a bispecific Ig-like molecule such as a bispecific antibody. In other embodiments, the Ig-like molecule is a monospecific Ig-like molecule such as a monospecific antibody. In a mixture of at least two different Ig-like molecules obtained by the method according to the invention, provided by a preferred embodiment, 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 is a heterodimeric Ig-like molecule obtained by the method according to the invention, wherein the heterodimeric Ig-like molecule binds to different epitopes on the same antigen and / or different epitopes on different antigens. The advantages and preferred uses of this mixture and the antibody have been described above.

[0183] In a mixture of at least two different Ig-like molecules obtained by the method according to the invention, provided by the invention, the at least two different Ig-like molecules comprise at least one heterodimeric Ig-like molecule. In one embodiment, the two of the at least two different Ig-like molecules are heterodimeric Ig-like molecules.

[0184] A further preferred embodiment provides a heterodimeric antibody comprising two CH3 domains, one of the two CH3 domains having the amino acid substitutions L351D and L368E, and the other of the two CH3 domains having the amino acid substitutions T366K and L351K. These amino acid substitutions are a 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 variant", "DEKK pair", "DEKK engineered CH3 domain", "DEKK", or other names that refer 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] The present invention also provides a pharmaceutical composition comprising a (heterodimeric) Ig-like molecule obtained by any method according to the present invention, or a mixture of at least two Ig-like molecules. The (heterodimeric) Ig-like molecule or the at least two Ig-like molecules according to the present invention are preferably antibodies. The pharmaceutical composition comprises the (heterodimeric) Ig-like molecule, a mixture comprising a monospecific or bispecific Ig-like molecule or a combination of monospecific and bispecific Ig-like molecules.

[0187] In addition, the pharmaceutical composition according to the present invention comprises a pharmaceutically acceptable carrier. As used herein, such a "pharmaceutically acceptable carrier" is any and all solvents, salts, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and agents that delay absorption, and physiologically miscible substances similar thereto, etc. Depending on the route of administration (e.g., intravenous, subcutaneous, intra-articular, etc.), the Ig-like molecule can be coated with substances that protect the Ig-like molecule from the action of acids and other natural conditions that can inactivate the Ig-like molecule.

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

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

[0190] The present invention is further illustrated by the following examples. These examples do not limit the present invention and are shown merely to clarify the present invention.

Brief Description of the Drawings

[0191]

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Mode for Carrying Out the Invention

[0192] (Example 1: Amino acid substitutions to generate various different CH3 domains) To selectively promote or inhibit the pairing of Ig-like molecules with CH3 domains and obtain a wide variety of different Ig-like molecules with CH3 domains, many amino acid substitutions known to promote heterodimer formation and many other amino acid substitutions not previously reported or tried but selected to promote homodimer formation were introduced into the construct vector (construct vector MV1057; Figure 1A).

[0193] The construct vector MV1057 comprises a nucleic acid sequence encoding the Fc portion of a normal wild-type IgG1 as described in Figure 2. Table 1 is a list of the amino acid substitutions introduced into this wild-type Fc, resulting in a series of seven constructs. All constructs were created by Geneart. Constructs 1, 2 and 3, or their alternatives, have previously been reported to promote heterodimerization (European Patent Application Publication No. 01870459, International Publication No. 2009 / 089004). There are also reports on constructs 6 and 7 (International Publication No. 98 / 50431). Constructs 4 and 5 are new and are designed to promote homodimers.

[0194]

Table 1

[0195] (Example 2: Cloning VH into constructs with CH3 mutations) For cloning into these constructs, several antibody VH regions with known specificities and the ability to bind to a known human IGKV1-39 light chain were used. As described above, all CH3 mutants can be used with other antibody domains to form full-length bispecific or monospecific antibodies. The specificity of the antibody determined by the VH / VL combination does not affect the dimerization behavior of the heavy chain induced by the CH3 domain. Model VH / VL combinations, i.e., combinations where all light chains are based on the human germline IGKV1-39 and the VH varies, are used throughout this study.

[0196] Figure 3 shows the full sequences and specificities of the antibody VH regions used throughout this study. The MF codes are internal designations for various VHs from Merus. For example, VH MF1337 is specific for tetanus toxoid, MF1025 for porcine butyroglobulin, and MF1122 for bovine fibrinogen.

[0197] The VH region in the phage display vector MV1043 (Figure 1B) is cleaved with the restriction enzymes SfiI and BstEII (New England Biolabs / cat# R0123L and R0162L / following the manufacturer's instructions), and the VH fragment is excised from this vector. By standard methods (following the manufacturer's instructions), the vector MV1057 is cleaved with SfiI and BstEII.

[0198] The fragment and vector are purified on a gel (Promega / cat# V3125 / following the manufacturer's instructions), and the cleaved vector and VH gene insert are isolated. Both are ligated together, and then the ligated nucleic acid is transformed into E. coli DH5α (Invitrogen / cat# 12297 - 016) (following the manufacturer's instructions). The next day, a single colony is 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 HEK293T cells with the recloned VH variants and various plasmids encoding the common light chain human IGKV1-39 is performed by standard procedures (de Kruif et al Biotech Bioeng. 2010) such that IgG can be expressed. After transfection, the expression level of IgG in the supernatant is measured by the ForteBIO Octet-QK system. This system is based on Bio-Layer Interferometry (BLI) and enables real-time quantification and dynamic characterization of biomolecular interactions. For details, see www.fortebio.com. If an expression level exceeding 5 μg / ml is measured, IgG is purified using protein A affinity purification.

[0200] (Example 4: Purification of IgG) After purifying the culture supernatant using a protein A column (GE Healthcare / cat# 11-0034-95 / following the manufacturer's instructions), it is eluted with 0.1 M citric acid buffer at pH 3.0 and immediately neutralized with an equal volume of 1.0 M Tris-HCL at pH 8.0 or directly rebuffered to PBS using a desalting column. Alternatively, IgG may 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 evaluate the binding activity to the antigen. Antigen-capture ELISA is performed to demonstrate the binding activity of the bispecific antibody. Biotinylated second antigen is used for detection of 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 according to standard procedures. Also, the proteins on the gel were stained with colloidal blue reagent (PageBlue™ protein staining solution / Fermentas / cat# RO571).

[0203] (Example 7: Enzymatic deglycosylation of IgG1) Since the glycosylation of IgG is heterogeneous, deglycosylation was performed so that one product would have a distinct single mass for suitable use in mass spectrometry analysis. One unit of N-glycosidase F (PNGase F; Roche Diagnostics, Mannheim, Germany) was administered to 10 μg of IgG1 and cultured 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 re-buffered with PBS. A similar buffer exchange procedure was performed to remove the separated glycan chains, and it was exchanged to 150 mM ammonium acetate at pH 7.5. The filter was rinsed with 200 μl of 150 mM ammonium acetate at pH 7.5 for 12 minutes at 11,000 rpm and 4°C. After being rinsed, 50 μl of deglycosylated IgG was administered to the filter and 450 μl of 150 mM ammonium acetate at pH 7.5 was added. Then, centrifugation was performed again for 12 minutes at 11,000 rpm and 4°C. Fresh 150 mM ammonium acetate buffer at pH 7.5 was added each time to make the total volume 500 μl, and centrifugation was repeated a total of 5 times. After the last centrifugation step, approximately 25 μl of the remaining deglycosylated IgG1, which had been buffer-exchanged, was recovered, 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 a purified mixture of IgG and to determine in what ratios these IgG species are present. Briefly, 2 - 3 μl of 150 mM ammonium acetate at pH 7.5 containing IgG at a concentration of 1 μM was loaded into a home - made gold - plated borosilicate capillary (using a Sutter P - 97 puller [Sutter Instruments Co., Novato, CA, USA] and an Edwards Scancoat six sputter coater [Edwards Laboratories, Milpitas, CA, USA]) and analyzed on an LCT1 mass analyzer (Waters Corp., Milford, MA, USA) that was tuned 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 a higher resolution “signal - to - noise” ratio was required. The source backing pressure was increased to approximately 7.5 mbar to promote collision cooling. To measure the denatured state of IgG1, the protein was sprayed at a concentration of 1 μM in 5% formic acid.

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

[0206] (Example 10: Mixture of two or three monospecific antibodies from a single cell) VH regions of several antibodies with known specificities and known human IGKV1-39 light chain binding ability (Figure 3) were recloned into the wild-type construct vector MV1057, or construct 4 or construct 5 in Table 1, resulting in vectors I-III (Table 2). Nucleic acid sequences encoding Ig heavy chains with different CH3 regions and different VH specificities, and a common human light chain, respectively, contained in vectors I, II, and III were then transfected into cells. The transfection was either done 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 is a list and result of the transfection.

[0207] [Table 2]

[0208] [Table 3]

[0209] From cells transfected with either vector I, II, or III by transfections A, G, and H, only homodimer formation of the bivalent monospecific AA, BB, or CC was obtained (Figure 4). For transfection A, this was predicted and has been demonstrated previously. For the first time, homodimerization of Ig heavy chains with engineered CH3 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) is reported (transfections G and H).

[0210] Next, co-expression experiments of two vectors in single cells were conducted. Interestingly, as shown by transfections M and N, when wild-type and engineered Ig heavy chains with CH3 were co-expressed together with a common light chain in single cells, there was no presence of unwanted bispecific antibodies, and only about 4-5% of "other molecules" were present in the mixture, resulting in a mixture of two monospecific antibodies. "Other molecules" are defined as all molecules that do not have the mass of intact IgG and include half-molecules consisting of a single heavy and light chain pair. The important point is that the "other" category does not include bispecific products.

[0211] In transfection M, the vector DNAs were transfected at equal ratios, and the ratio of AA:BB was approximately 1:1. However, in transfection N, the ratio of AA:CC was approximately 10:1. Therefore, this transfection was repeated after adjusting the DNA ratio (transfection U). In fact, when the ratio of 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, without unwanted by-products (i.e., no large amounts of AC or half-molecules A or C), it is possible to express two different, substantially non-mixed, monospecific antibodies in single cells (Figure 5). The novel CH3 modifications of constructs 4 and 5 are substantially different from the wild-type CH3 so that heterodimer formation between wild-type and construct 4, or wild-type and construct 5, does not occur. This aspect is advantageous for the application to the large-scale production of mixtures of monospecific antibodies from single cells.

[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 single-specificity antibodies without the presence of additional unwanted species. If the modification of CH3 in construct 4 is substantially different from the modification of CH3 in construct 5, it is inferred that hetero-dimerization 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 single-specificity antibodies.

[0213] Indeed, this result was observed. When three different Ig heavy chains designed to form homo-dimers rather than hetero-dimers were expressed in a single cell with a common light chain, it was found that a mixture of three pure single-specificity antibodies was obtained without other contamination in the mixture (transfection O) (Figure 6). As is clearly shown from Table 3, even when the vector DNA was made to be in the same ratio in transfection O, the ratio of antibodies AA:BB:CC was not obtained at 1:1:1. By varying the ratio of vector DNA for transfection (1:1:10, transfection V), it was shown that the ratio of AA:BB:CC in the mixture could be manipulated to the desired ratio. From the above, as shown by these experiments, two or three substantially pure single-specificity antibodies can be produced in a single cell without unwanted by-products. This is of great advantage for the large-scale production of mixtures of therapeutic single-specificity antibodies.

[0214] (Example 11: Mixture of Two Bispecific Antibodies from a Single Cell) The production of a single bispecific antibody using a CH3-engineered heavy chain 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 region of an antibody having a known specificity and the ability to bind to the known human IGKV1-39 light chain (Figure 3) was recloned into a vector containing constructs 1-3 or 6-7 of Table 1, resulting in vectors IV-X (Table 4). Nucleic acid sequences encoding Ig heavy chains with different CH3 regions and different VH specificities, and a common human light chain, were each contained in vectors IV-X, which were then transfected into cells. The transfection was done either alone to show interference with the formation of intact single-specificity antibodies, or in combination with other construct vectors to obtain bispecific antibodies or a mixture of two bispecific antibodies. Table 5 is a list and result of the transfection.

[0215]

Table 4

[0216]

Table 5

[0217] When the CH3 encoded by constructs 1 and 2, which have engineered Ig heavy chains, are expressed in a single cell, they have been shown to maintain the ability to form homodimers (WO 2009 / 089004). However, in a further report of WO 2009 / 089004, CH3 domains engineered to have pairs of triple-charged mutations, such as construct 3, are unable to form homodimers when expressed alone. In this study, these findings were only partially confirmed. In fact, in transfections B, C, and D, in addition to a high proportion of unpaired half-molecules, the presence of full-length IgG was shown. This indicates 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. This indicates that the triple-charge mutations in construct 3 do not completely abolish homodimerization. The "knob" and "hole" CH3 mutants of constructs 6 and 7 were further shown to form homodimers (18% for the "knob-knob" homodimer and 42% for the "hole-hole" homodimer).

[0218] When co-expressing a second CH3 mutant for heterodimer formation, a CH3 mutant in which homodimerization is completely inhibited when expressed alone is preferred to inhibit or minimize unwanted by-products (homodimers).

[0219] Interestingly, this experiment demonstrated for the first time that it is possible to express a mixture of bispecific antibodies in a single cell with substantially no homodimers in the mixture. In transfections K and L, the predicted bispecific species BC+AB were actually obtained (38% + 47% in transfection K and 16% + 60% in transfection L). A relatively high percentage of unwanted half-molecules was observed in both transfections (15% half-molecule A + half-molecule C in transfection K and 24% half-molecule A + half-molecule C in transfection L). The half-molecules that still exist at a relatively high percentage are due to the lack of a balanced expression of the heavy chains of the compatible pairs, resulting in a low amount of the heavy chain of vector IV. Therefore, in transfections S and T, the ratio of vector DNA was adjusted to 2:1:1 and transfected again. As a result, equal amounts of IgG heavy chains consisting of compatible pairs were obtained, there was no presence of IgG half-molecules, and a pure mixture of bispecific IgG was obtained with only the presence of 3% homodimer BB. Ideally, this low percentage of contaminating monospecific products should be reduced substantially to zero. Therefore, it is desirable to find further CH3 mutants that can yield a mixture of bispecific antibodies with minimal presence of contaminating monospecific antibodies.

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

[0221] (Example 12: Various Mixtures) It was shown that the production of a mixture of two bispecific antibodies that recognize three epitopes from a single cell, or the production of a mixture of two or three monospecific antibodies from a single cell, is technically feasible. We then explored the feasibility of the controlled production of various other mixtures.

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

[0223]

Table 6

[0224] Theoretically, the production of all mixtures is feasible, but from previous other studies, it is known that the large-scale production of the previously engineered knob-into-hole variants has been hampered due to stability problems. Therefore, the mixtures resulting from transfections ZA, ZB, ZL, ZM and ZN are predicted to have problems when moving to large-scale production.

[0225] That is, with the set of constructs in Table 1, it may not be possible to produce all the theoretical mixtures by mass production from a single cell. The reason is that the knob-into-hole mutants have been reported to be unstable, and it cannot be excluded that the CH3 domains with "knob" or "hole" dimerize with either of the charged mutants or the wild-type CH3 domain. Therefore, in order to co-express in a single cell, it is desirable to design new CH3 mutants that selectively form only homodimers or heterodimers and do not homodimerize or heterodimerize with constructs 1-5 in Table 1.

[0226] (Example 13: Identification of Mutations in Novel Charge Pairs) The purpose of this study is to manipulate the CH3 region of IgG so that when different IgG heavy chains are co-expressed in a single cell, only heterodimers or only homodimers are produced as a result. Here, the newly engineered CH3 domain is prevented from homodimerizing or heterodimerizing with the known engineered CH3 domain or the wild-type CH3 domain. Therefore, as a first step to identify newly engineered CH3 domains that meet this criterion, many of the contact residues on the contact surface of the IgG CH3 domain were replaced one by one or as a group to examine whether repulsion between the same heavy chains due to electrostatic interaction - that is, a decrease in homodimer formation - occurs. The purpose is to obtain a list of residues that, when replaced with charged residues, cause repulsion between the same chains so that these mutations can be used to induce the formation of homo- and / or heterodimers when different IgG heavy chains are co-expressed. As a result, the resulting full-length IgG becomes stable and is produced at a high rate.

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

[0228] Samples are then analyzed by SDS-PAGE under non-reducing conditions to identify pairs in which dimer formation is decreased by looking for bands at approximately 72 kD. All obtained pairs were screened for single mutations or combinations with other single mutations. The reason is that it is unclear whether the repulsive electrostatic interactions due to a single non-compatible pair are sufficient to yield an amount of half-molecules detectable by this method. These mutations are also used in combination.

[0229] According to Table 7, amino acid substitutions were introduced into the construct vector MV1057 by Geneart. Also, expression of the construct was performed by transfection into HEK293T cells according to standard procedures. The IgG expression level was measured by Octet. When production failed twice, the mutation was considered to inhibit expression and no further investigation of the mutation was performed.

[0230]

Table 7

[0231] The supernatant containing IgG at ≧5 μg / ml was analyzed by SDS-PAGE and purified using Protein A. The protein was stained using a colloidal blue reagent. The homodimer could be captured as a band of approximately 150 kD. The smaller band of approximately 75 kD indicated the presence of half-molecules (see negative controls: K392D, K409D). The blot is shown in Figure 7.

[0232] The results of SDS-PAGE were analyzed and scored and presented in Table 7, rightmost column. Many residues including Q347, S354, Y349, L351, K360, T366, T394, and V397 are promising and should be further tested in combination. The selection here considered both a high score in the inhibition of homodimer formation and the availability of modifiable contact residues that would not pose problems in relation to other non-complementary charges. For example, the F405 residue and the Y407 residue are known to have multiple interactions at the CH3-CH3 contact surface, including interactions with already charged residues. Introducing multiple charge mutations among these interacting residues (see Table A) could be problematic.

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

[0234]

Table 8

[0235]

Table 9

[0236]

Table 10

[0237] Combinations of CH3 variants were expressed and analyzed by SDS-PAGE (data not shown) and native mass spectrometry (MS). The results were summarized in Table 10. By ZO transfection, the highest proportion of heterodimers in the mixture was obtained (69% AC). Interestingly, in ZO transfection, no AA homodimers were present, while the CC homodimers contained a small proportion (7%). According to mass spectrometry, the proteins remaining in the mixture consisted of half-molecules of A, which is considered to be the result of unbalanced expression of A and C heavy chains. The raw MS data from the transfection sample ZO are shown in Figure 8. Surprisingly, a significant amount of bispecific product was obtained by transfection ZO, while its inversion charge pair, transfection ZP (ZO is T366K / L351’D versus L351K / T366’D for ZP), could not obtain similar results, and only 52% of bispecific products were observed, and a significant amount of two homodimers were present (30% AA and 13% CC). In contrast, since the negatively charged D is structurally very similar to T, T366D is not strong enough to repel itself, so it is explained that T366D can still form homodimers, and this was actually observed.

[0238] It is predicted that similar proportions of bispecific antibodies (BsAbs) will result from slightly modified variants of the newly discovered T366K / L351’D pair (e.g., by testing all modifications including the new constructs T366R and L351E).

[0239] (Example 14: Design of new CH3 mutations for inducing efficient heterodimerization by HADDOCK) As described in Example 13, the newly discovered charge pair T366K / L351’D increases the proportion of heterodimers in the mixture (69%), while contaminating the mixture with a small proportion of unwanted CC homodimers (7%) (L351D / L351’D) and a significant proportion of half-molecules A (24%). In this example, an in silico approach was used to obtain further insights into the amino acid residues involved in the interaction at the CH3 contact surface, test complementary substitutions facing each other in the CH3 region, and find a new CH3 pair containing complementary substitutions that inhibit the 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-based flexible docking approach for modeling biomolecular complexes. Unlike first-principles docking methods, HADDOCK encodes information on the protein contact surfaces identified or predicted within ambiguous interaction restraints (AIRs) and guides the docking process (de Vries et al., 2010). The input to the HADDOCK web server consists of a protein structure file such as a crystal structure, an NMR structure cluster, or a structure model. After docking or fine-tuning, HADDOCK returns a so-called HADDOCK score. The HADDOCK score is a weighted average of van der Waals energy, electrostatic energy, buried surface area, and desolvation energy. Although the HADDOCK score is often difficult to directly convert into experimental data, it is interpreted as an indicator of binding energy or affinity. In addition to this, HADDOCK provides the structure files of the "top 4" structures from the results of the docking calculations. These structure files can be downloaded and visualized, enabling a detailed analysis of the interactions of individual residues.

[0241] In this example, the interaction between the CH3 domains of the IgG1 heavy chain is studied. A structure starting from the Fc region of IgG (structure 1L6X) with a high-resolution crystal structure is used (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=1l6x ; Idusogie, E.E. et al., J.I. 2000(164)4178-4184).

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

[0243] The output of the HADDOCK score consists of the calculated energy, the HADDOCK score (a weighted average of several energies), and four structure files corresponding to the four minimum energy structures found by the program. The HADDOCK score is used to compare different structures. Other energies are only used to obtain an indication of what is happening in the structure (e.g., good electrostatic interactions, less buried surface, high van der Waals energy). The lower the HADDOCK score, the better. For each mutant pair, the scores of the AA, AB, and BB dimers are calculated.

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

[0245]

Table 11

[0246] For the two wild-type CH3 domains, since the CH3 regions of A and B are homologous, the HADDOCK scores are identical for AA, AB, and BB. In many other cases, as predicted, the AB pair had the lowest score. For the T366K / L351D pair, the BB score was slightly better than the AB score (-210.6 vs. -212.5). However, this difference is within the range of computational error. Using HADDOCK, the structures of the heterodimers of these pairs were visualized. For example, the construct combinations 1-2, 1-1, and 2-2 are shown in Figure 10. From these visualizations, it is clear that salt bridges are formed in the heterodimer (left panel of Figure 10A), and electrostatic repulsion occurs between residues of the same chain (central and right panels of Figure 10B and C). The higher HADDOCK scores for the homodimers are explained by the electrostatic repulsion of the mutated contact residues. These residues avoid bending towards each other and do not interact with residues of the other chain, resulting in a decrease in 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. On the other hand, the AA homodimer of T366K / T366’K was hardly detectable in the mixture, and the T366K monomer A was 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. Therefore, the formation of this homodimer is less energetically favorable.

[0248] (Example 15: 366 / 351 Mutation) In Example 13, a hypothesis is proposed that by a method different from the pair of mutant charges of T366K / L351’D, it can also be designed to obtain similar results regarding the proportion of bispecific antibodies in the mixture. The different method may include substitutions of T366R, T366D, T366E, L351E, L351K, and L351R. The proportion of the CC homodimer of L351D / L351’D can be reduced by generating mutants of the 366 / 351 pair. All possible pairs of mutations 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 variations, the AA homodimer was found to have a higher HADDOCK score than the AB heterodimer, but the BB homodimer was equally preferred to the AB heterodimer. Residue 351 is known to be "next to" the same residue on the other chain. That is, residue 351 of chain A pairs with residue 351 of chain B at the CH3-CH3 contact surface. When the BB dimer is formed, there is little negative impact due to the same charge. Looking at the structure of L351D / L351’D for explanation, aspartic acids bend away from each other and avoid each other, and there is at least the stabilizing effect from the naturally occurring arginine at position 355, and further the stabilization of the negative charge by the naturally occurring serine at position 354 (see Figure 12A). Mutating these residues (S354A and R355D) results in little improvement. From Figure 12B, it is clear that the backbone hydrogen of A354 is responsible for the stabilization of the homodimer. From this series, the T366R / L351’E pair is considered to have the minimum HADDOCK score for the bispecific molecule and is the most suitable.

[0251] (Example 16: Mutations around T366K / L351’D) In a series of HADDOCK analyses in this example, pairs of T366K / L351’D or T366K / L351’E were used as starting structures. To identify additional mutations that could further increase the predicted ratios of bispecificity for these A and B chains, mutations were added to the B chain and HADDOCK scores and energies were calculated. When the CH3 domain structure was examined using a viewer (YASARA, www.yasara.org) for visualization of protein structures at the molecular level, the distances between individual residues could be calculated. During that examination, two residues, Y349 and L368, were observed to be adjacent residues that could make positive or negative contributions to the dimer interaction. In this example, the effects of these mutations - in addition to the L351 mutation - on homodimer and heterodimer formation were examined (see Figure 13). Both residues increased the stability of the heterodimer (lower HADDOCK score) and destabilized the BB dimer (higher HADDOCK score). Glutamic acid (E) at positions 349 and 368 was suggested to be more suitable than aspartic acid (D). Therefore, the introduction of a second amino acid mutation in the B chain already having an amino acid substitution at position 351 was suggested to make heterodimerization preferable.

[0252] In the next set of HADDOCK analyses, the T366K / L351’D pair was again used as the starting structure. Additional mutations were introduced into the A chain with the T366K substitution to the substitutions in the B chain (i.e., Y349D / E and L368E) that led to further increases in heterodimerization. As shown in Figure 14, there are several mutations that are considered suitable for the formation of dual-specific heterodimers. In the T366K-L351K / L351’D-Y349’D pair, all four mutated residues are involved in 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 -214.2 for T366K / L351’E-L368’E. This can be explained by the hydrogen bond interaction at K in position 351 (see Figure 15). The T366K-L351K / L351’D-Y349’D pair can be further improved by the R355’D mutation in the B chain. This increases the HADDOCK score of BB, but also slightly increases the HADDOCK score of AB. From the above, 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, a higher amount of dual-specific heterodimers in the sample will be obtained.

[0253] As is clear from Figure 11, it is speculated that changing position 366 to R instead of K is more effective in inducing heterodimerization. Therefore, this time, instead of T366K, T366R was used in the A chain, and several HADDOCK analyses shown in Figure 13 were repeated. It was shown that it is not preferable to combine the R366 in the A chain with double mutations in the B chain (Figure 16). This is because the size of this residue is large and, even if all salt bridges with R366 are present in the structure, it interferes with the interactions of other contact surfaces. Furthermore, the HADDOCK score of the AA homodimer is lower for R366 than for K366. This is also an unfavorable contribution to heterodimer formation. Therefore, further HADDOCK analysis using R366 at the contact surface was discontinued.

[0254] From the predictions by HADDOCK, pairs that achieved a total of 14 optimal scores were selected (see Table 13 and Figure 17). In some pairs, an R355D substitution was included to remove the effect of the natural stabilization of 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 proportion of the bispecific component and a lower proportion of the homodimer component. These pairs with optimal scores were selected for production and further analysis was performed. Additionally, constructs T366R and L351E were also generated. A list of the constructs created and the constructs used for recloning antibody VH regions with the ability to bind to known specificities and a known human IGKV1-39 light chain is shown in Table 14.

[0257] The expression levels of IgG containing individual constructs were reported in Example 13 above and were also repeated for the constructs listed in Table 14. The purpose is to evaluate which constructs homodimerize in the absence of a compatible partner for heterodimerization. Ideally, a high proportion of half-molecules and a low proportion of homodimers should be formed. As a control group, constructs containing charge mutations reported so far and constructs containing knob-in-hole mutations reported so far were also used for the expression of whole IgG by recombinant cells. The supernatant purified by Protein A was analyzed by SDS-PAGE, and the results are shown in Table 14 together with the scores.

[0258] [Table 14]

[0259] The results of co-expression of a common light chain with two different heavy chains retaining the amino acid substitutions of the constructs shown in Table 14, or a heavy chain retaining the amino acid substitutions of the previous constructs, are shown in Table 15. Expression of two different heavy chains each with amino acid substitutions T366K and L351’D:L368’E resulted in the absence of homodimers AA or BB, and approximately 87% of bispecific heterodimer AB could be obtained in the mixture (Combination No. 3 in Table 15). Half-molecules (Half-molecule A) containing approximately 12% of the T366K substitution were observed. Furthermore, when an additional amino acid substitution L351K was introduced into the first heavy chain, it was found that the proportion of bispecific heterodimer AB increased. For example, each with amino acid substitutions T366K:L351K and L351’D:L368’E Co-expression of two different heavy chains resulted in approximately 92% bispecific heterodimer AB being obtained, while homodimers AA and BB were substantially absent from the mixture (Combination No. 12 in Table 15).

[0260] Combinations 10 and 11 also result in a favorable distribution of a high proportion of heterodimers and homodimers that are substantially absent. The absence of homodimers is advantageous because the fraction containing intact IgG molecules is composed only of heterodimer AB. For purification and subsequent therapeutic applications, half-molecules can be removed by standard approaches such as size exclusion chromatography. Known charge variants and knob-into-hole variants do not remove "contaminating" homodimeric antibodies. However, applying these newly identified charge variants to the production process of bispecific antibodies is advantageous.

[0261] In addition, the T366K / L351’D:L368’E and T366K:L351K / L351’D:L368’E charge pairs have further advantages over the E356K:D399K / K392’D:K409’D and E356K:D399K / K392’D:K409’D:K439’D charge inversion pairs described previously. That is, the charge variants described previously are based on the inversion of the charges originally present within the CH3-CH3 contact surface, while the newly identified charge variants add additional charge pairs (charge-charge interactions) to the CH3-CH3 contact surface. The introduction of additional charge pairs at the CH3-CH3 contact surface further increases the stability of the contact surface, thereby further increasing the stability of the intact antibody. The same applies to the mutations used in combination numbers 4, 5, 6, 9, 10, and 11. This leads to the preferred ratio of bispecific heterodimers where the AA and BB homodimers are present in the mixture at a very low ratio only.

[0262]

Table 15

[0263] (Native MS) Native MS was performed on all bispecific 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 method based on peak area is scientifically the more correct analytical method, but since all previous analyses in other studies have been performed using peak height, both methods were included in the analysis for comparison purposes. The difference between the two methods was within the range of measurement error, so only the peak area values were used for subsequent measurements.

[0264] Two typical spectra are shown in Figure 18. The summary of the results is shown graphically in Figure 19. The numerical values can be found in Table 15. In approximately half of the samples, the total contamination of monospecific IgG was less than 5%, and in only three cases did it exceed 10%. On the other hand, it is predicted that approximately 50% monospecific IgG will be found in the mixture for wild-type IgG.

[0265] For further analysis, 10 combinations of two different heavy chains were selected from Table 15. These 10 combinations include combinations 1, 2, 3, 4, 5, 6, 9, 10, 11, and 12 (Table 15). These 10 selections were made based not only on the low proportion of homodimers present in the mixture derived by nMS, but also on the overall physicochemical properties including production yield, SDS-PAGE, and the number of mutations in the CH3 domain.

[0266] (Example 18: Analysis of IgG Stability) In this study, for a series of pairs of CH3 mutations when a high proportion of bispecific heterodimers and a very small amount (<5%) of parental IgG were obtained in the intact IgG fraction, the stability of the Fc portion of the IgG molecule was further analyzed. The mutated CH3 domains used to promote heterodimerization of the heavy chains can have an unexpected destabilizing effect on the Fc region of IgG. This can result in undesirable properties such as a decrease in the in vivo half-life, a decrease in effector function, and / or an increase in immunogenicity.

[0267] For the newly identified charge pairs, they were compared with the wild-type bispecific molecule and the bispecific molecules containing the charge mutations identified so far (chain A with construct 1 and chain B with construct 2). Since all the bispecific molecules in this study contain the same heavy and light chain variable regions, it is ensured that the observed effects are due to the mutations in the Fc portion of the molecule and not due to differences in the variable regions.

[0268] A series of stability studies were conducted on these bispecific molecules. These studies include spectroscopic (UV-Vis light absorption, fluorescence, and light scattering) and microscopic (observation by optical and fluorescence microscopy with Nile red staining) analyses that provide information on the aggregation state of the CH3 variants.

[0269] The UV-Vis absorption spectrum is recorded at 25 °C using a Cary 300 Bio spectrophotometer with two monochromators in double-beam mode. The spectrum is monitored between 250 and 400 nm with a path length of 1 cm. Information on the aggregation state of IgG is provided by the absorption at 320 nm and longer wavelengths.

[0270] The intrinsic fluorescence spectrum is monitored at 25 °C using a FluoroMax fluorimeter. The fluorescence method is optimized as appropriate. Fluorescence emission provides information on conformation and aggregation characteristics.

[0271] The 90° light scattering spectrum is monitored at 25 °C using a FluoroMax fluorimeter with an integration time of 0.01 s and a synchronous scan from 400 nm to 750 nm (λ em =λ ex ). The excitation and emission slits are optimized as appropriate. For example, in right-angle light scattering, it is possible to distinguish whether 5% dimers are present in the IgG sample.

[0272] In fluorescence microscopy observation with Nile red staining, immediately before measurement, Nile red contained in ethanol is added to the sample. The sample is filled into a microscope slide and analyzed by a fluorescence microscope. Particles are counted, but the minimum particle size observed by fluorescence microscopy is about 0.5 μm.

[0273] Stresses on proteins such as temperature, pH, mechanical stress, or denaturants cause conformational changes (e.g., unfolding) and / or aggregation. So far, it has been reported that bispecific antibodies with engineered charges have a lower melting temperature of the modified CH3 (Gunasekaran 2010). Therefore, these studies aim to distinguish the novel charge variants of the present invention from the already existing and known charge variants. Stresses on proteins such as temperature, pH, mechanical stress, or denaturants cause conformational changes (e.g., unfolding) and / or aggregation. So far, it has been reported that bispecific antibodies with engineered charges have a lower melting temperature of the modified CH3 (Gunasekaran 2010). Therefore, these studies aim to distinguish the novel charge variants of the present invention from the already existing and known charge variants.

[0274] Using a Protein A biosensor and FcRn to IgG, the thermal stability was studied by Octet. To examine the thermal stability of IgG with engineered CH3, a PCR machine was used to incubate the samples at 4, 50, 55, 60, 65, 70, and 75 °C for 1 hour at a concentration of 100 μg / ml (PBS as solvent). After that, the samples were slowly cooled to 25 °C in 15 minutes, kept at this temperature for 2 hours, and then stored at 4 °C until the next day. The precipitated antibody was removed by centrifugation, and the total IgG concentration of the water-soluble antibody was derived by Octet using an Octet Protein A biosensor (1 / 10 PBS dilution).

[0275] An assay was studied to measure the binding of engineered IgG to FcRn using Octet. After binding the IgG light chain to the sensor using a Protein L biosensor, it was incubated with FcRn in solution. Alternatively, it may be incubated with the target IgG after binding to the His-tagged FcRn protein using an Anti-Penta-HIS biosensor. These methods may be more sensitive than the Protein A biosensor and may also be used for thermal stability studies.

[0276] All samples were also subjected to analysis of stability under serum. Briefly, the (engineered) IgG samples were incubated at 37 °C in human serum, and the control samples were kept at 4 °C. After 1, 2, 3, and 4 weeks, the samples were centrifuged to remove the precipitated IgG. Then, the samples were added in an antigen-specific ELISA, and the relative amount of functional IgG was determined. The purified control antibody was rapidly administered to human serum and used as a reference group.

[0277] (Example 19: Analysis of Stability) In previous experiments, a high proportion of bispecific antibodies were obtained by co-expression of two different heavy chains with an engineered CH3 and a common light chain (Example 17).

[0278] Eight combinations of two different heavy chains were selected from Table 15 and further analyzed. These eight combinations included combinations 3, 4, 5, 6, 9, 10, 11 and 12 (Table 15). In this study, the focus was on the stability of the Fc portion of IgG, and these eight combinations were analyzed. The control group included wild-type bispecific molecules (i.e., without CH3 mutations) and / or previously reported charge mutations of CH3. Note that for wild-type bispecific molecules, two heavy chains and a common light chain were co-expressed without a means to selectively induce heterodimers. Thus, these "wild-type bispecific molecules" represent a mixture of AA, AB and BB. Since all bispecific molecules in this study retain the same heavy and light chain combinations, it is ensured that the observed effects are due to mutations in the Fc portion of the molecule and not due to changes in the Fab portion.

[0279] As a hypothesis, mutant pairs used to promote heterodimeric pairing of two different heavy chains may be associated with unanticipated structural or other destabilizing effects on the IgG Fc region. This could later result in undesirable problems that could impede further clinical development, such as a decrease in the in vivo half-life, a decrease in effector function, and / or an increase in immunogenicity due to the presence of these mutations.

[0280] (Thermal stability) Applying stress such as an increase or decrease in temperature causes changes in protein conformation (e.g., unfolding) and / or aggregation. To examine the thermal stability of CH3-engineered IgG, combinations 3-6 and 9-12 (Table 15), together with wild-type bispecific molecules and bispecific molecules obtained using constructs 1 and 2 (the combination of E356K:D399K / K392D’:K409D’, also called the “charge reversal” pair), were incubated at 4, 60, 62.5, 65, 67.5, 70, and 72.5 °C for 1 hour at a concentration of 100 μg / ml (PBS as the solvent) using a PCR device. Subsequently, the samples were slowly cooled to 25 °C over 15 minutes, maintained at this temperature for 2 hours, and then stored at 4 °C overnight. The precipitated antibody was removed by centrifugation (18,000 rpm; 4 °C, 20 minutes), and the total IgG concentration of the water-soluble antibody was derived using an Octet with a protein A biosensor (diluted 1 / 10 in PBS).

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

[0282] (Stability to freeze-thaw) To examine the stability of CH3-engineered IgG upon 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 (combination of E356K:D399K / K392D’:K409D’ (charge-reversed pair)) were subjected to 10 freeze-thaw cycles. In each cycle, the sample was placed at -80 °C for at least 15 minutes until completely frozen and then thawed at room temperature. Once completely thawed, this freeze-thaw cycle was repeated. After 10 freeze-thaw cycles, the precipitated antibody was removed by centrifugation (18,000 rpm; 4 °C, 20 minutes), and the total IgG concentration of the water-soluble antibody was derived using an Octet with 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 charge-reversed CH3-engineered bispecific antibody, which served as a control, was observed to have slightly reduced stability compared to the wild-type bispecific molecule. In contrast, the bispecific molecules from combinations 3, 4, and 9 were considered to have slightly improved stability compared to the wild-type bispecific molecule. From the above, it was concluded that no significant stability issues were raised for CH3-engineered variants under the harsh conditions of freeze-thaw cycles. It was concluded.

[0284] (in vitro serum stability) To examine the stability of CH3-engineered IgG in serum maintained at 37 °C, bispecific molecules from combinations 3-6 and 9-12 (Table 15), wild-type bispecific molecules, and charge-reversed bispecific molecules were incubated under 10% human serum at 37 °C. Control samples were kept at 4 °C. After 1, 2, or 5 days, the precipitated antibody was removed by centrifugation. The sample was then added in a fibrinogen-specific ELISA to derive the relative amount of functional IgG. The purified control antibody was rapidly administered to human serum and used as a reference group.

[0285] According to the fibrinogen ELISA data, all samples were very stable even after 5 days at 37°C in 10% human serum. Bispecific molecules with lower IgG concentrations from combinations 4 and 5 were slightly less stable, especially at T = 1 and T = 2. However, the difference was minimal at the end of this experiment (Figure 22).

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

[0287] All IgG were diluted to 0.2 mg / ml and several stress conditions (2 days at 50°C, 2 weeks at 40°C, 5 freeze - thaw cycles) were applied. This was to enable discrimination between different samples. Note that these high stress levels are such that one of the parental antibodies (the BB - side parent that holds two 1122Fabs) used in all bispecific molecules becomes unstable. Protein aggregation of this protein was detected by UV absorption under the condition of 2 days at 50°C. As suggested by this, it may not be possible to distinguish between the instability of Fab and CH3 in the bispecific molecule under this stress condition. Therefore, the data from the incubation at 50°C should be treated with caution and.

[0288] An overview of the results is shown in Table 16. The analytical methods used included the following. - Fluorescence microscopy with Nile red (“Nile red particles” in Table 16); to observe the amount of particles larger than 0.5 μm after adding the Nile red dye. -UV spectroscopic analysis at 350 nm (“UV350 nm”); changes in absorption at wavelengths longer than 320 nm provide information on the aggregation state of the protein. -90° light scattering at 400 nm (“LS400 nm”); a sensitive technique for observing changes in protein aggregation, such as the difference between monomeric and dimeric IgG. -Autofluorescence; the maximum wavelength and intensity of the fluorescence of the aromatic residues of the protein vary depending on the environment (e.g., unfolding). -1,8-ANS fluorescence spectroscopy; 1,8-ANS binds to a group of cations through electrostatic interactions via ion pair formation. Changes in protein structure and / or conformation are detected 。

[0289] (UV-Vis spectroscopic analysis) The UV-Vis absorption spectra were measured at 25 °C using different quartz cuvettes from Varian (e.g., a black low-dose Hellma cuvette with an optical path length of 1.0 cm and a 0.2 cm x 1.0 cm transparent Hellma cuvette) and a Cary 300 Bio spectrophotometer with a double beam and two monochromators. Using an optical path length of 1.0 cm, the spectra between 220 and 450 nm were monitored. Absorption near 280 nm provides information on the protein concentration. The region between 320 and 450 nm provides information on the aggregation state of the sample.

[0290] (90° light scattering) The 90° light scattering spectral method was developed to study protein aggregation. The method was performed as described in Capelle 2005 and Demeule 2007a. The 90° light scattering spectra were monitored at 25 °C using a FluoroMax fluorimeter (Spex, Instruments S.A., Inc. U.K.) with an integration time of 0.01 s by performing a synchronous scan (λem = λex) from 400 nm to 750 nm. Different slit settings were tried to obtain optimal conditions. After optimization, all measurements were performed using that slit setting.

[0291] (Steady-state fluorescence emission) The fluorescence emissions of tryptophan, tyrosine, and phenylalanine residues provide information on the local environment of these fluorophores. Changes or differences in hydrophilicity and / or rigidity are measured. Generally, a more hydrophobic and rigid environment leads to an increase in fluorescence intensity and a blue shift in the emission maximum value. Information on the state of the protein at that time is provided by autofluorescence spectroscopy, and changes in physical and chemical properties are monitored. More detailed information on tyrosine and tryptophan is described in Lakowicz's book (Lakowicz, 2006).

[0292] The fluorescence emission and excitation spectra were recorded at 25 °C in different quartz cuvettes. The 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) The Nile red staining method was developed to visualize protein aggregation. The staining was performed as described by Demeule et al., 2007b.

[0294] Microscopic observations were performed using a Leica DM RXE microscope (Leica Microsystems GmbH, Wetzlar, Germany) equipped with a mercury lamp. Images were acquired using a Sony NEX-5 camera and its firmware. The objective lenses were 10×, 20×, and 40×. In the microscopic study, the distance between the slide and the cover glass was fixed at 0.1 mm and used. The size of the 4×4 grid was 1 mm × 1 mm, corresponding to 0.1 μl.

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

[0296] 1,8-ANS does not fluoresce substantially in water and emits detectable fluorescence only when bound to a membrane (quantum yield ~0.25) or a protein (quantum yield ~0.7). Due to this property, 1,8-ANS is a highly sensitive indicator of protein folding, conformational changes, and other processes in which the exposure of this probe to water changes. Information on 1,8-ANS can be obtained from the Molecular Probes homepage at www.probes.com.

[0297] The fluorescence emission spectrum of 1,8-ANS was recorded using a FluoroMax fluorometer. A direct comparison of the fluorescence of 1,8-ANS between each IgG was not performed because each IgG has a different number of 1,8-ANS binding sites and thus cannot be compared. In principle, the smaller the fluorescence of 1,8-ANS, the fewer 1,8-ANS molecules are bound to the antibody. The changes in the fluorescence intensity and emission wavelength of 1,8-ANS due to stress were evaluated.

[0298]

Table 16

[0299] In summary, these data showed that the various IgG samples were remarkably stable. Severe stress conditions (e.g., 50 °C for 2 days) were required to produce a measurable difference between the tested samples. Under these conditions, the samples with combination numbers 9 and 10 seemed to aggregate more easily than the other samples.

[0300] The greatest factors that can distinguish the stability between proteins are freeze-thaw cycles and temperature rise. Considering the incubation at 50 °C, which is a very severe stress factor, two mutants, T366K / L351E, Y349E (combination number 4) and T366K, L351K / L351D, Y349E (combination number 11), are the most stable proteins in this group, followed by T366K, L351K / L351D, Y349D (combination number 10) and T366K, L351K / L351D, L368E (combination number 12) with a slight difference.

[0301] (Example 21: Experiment of performing native MS with varying ratios; transfection ratio from 1:5 to 5:1) To deepen the knowledge of the behavior of CH3-mutated IgG in the mixture when transfected at a biased ratio, especially for the combination of T366K:L351K / L351D’:L368E’ (hereinafter referred to as KK / DE or DEKK), more detailed experiments regarding the ratio were conducted.

[0302] The VH regions of the previously used antibodies, which have known specificities and the ability to bind to the known common light chain human IGKV1-39, were used to reclone into constructs 1, 2, 68, and 69, resulting in vectors I-V (Table 17). Nucleic acid sequences encoding Ig heavy chains with different CH3 regions and different antigen specificities, along with a common human light chain, were included in vectors I-V and transfected into cells at different transfection ratios shown in Table 18. The results are shown in Figure 23.

[0303]

Table 17

[0304]

Table 18

[0305] As shown in FIGS. 23A and B, in the DEKK combination mutation, when an excess amount of A or C is present (A or C is the "DE side", B is the "KK side"), AB or BC is formed, but in all cases, the excess amount of A or C exists as a mixture of both homodimers and half-molecules. However, when an excess amount of B is present (B is the "KK side", A or C is the "DE side"), a distinct difference is seen. AB or BC is still formed, but the excess amount of B does not substantially exist as a homodimer and only half-molecules are formed.

[0306] It should be noted that here too the ratio is measured by the height of the peak. For peaks detected at 2% or less, it is below the threshold that can be accurately measured by the nMS technique used here. Therefore, measurements less than 2% were considered within the range of the analysis noise level and were ignored.

[0307] It is noteworthy that when B is exceeded, only the proportion of half-molecule B increases. In particular, in the cases of the ratios of A:B being 1:3 and 1:5, the presence of the homodimer BB is absent and a high proportion of half-molecule B is observed (FIGS. 23A and 23B), indicating that homodimers are less likely to occur with the KK-side CH3 mutation. The absence of homodimers is a decisive advantage. The reason is that when the "KK side" of the DEKK combination is selected to add specificity, there is a possibility that the previously known adverse effects may occur when present as a homodimer (for example, cMET or CD3 antibodies are known to have undesirable adverse side effects when present in a pharmaceutical composition as a bivalent homodimer).

[0308] The observation results showing different ratios of DE:KK are in contrast to the control charge inversion CH3 mutations in vectors IV and V. As shown in Figure 23C, in the mutation of the combination of E356K:D399K / K392D’:K409D’, when A is present in excess (A is on the “K392D:K409D side”), in all cases, the excess amount of A exists as a mixture of both homodimers and half-molecules. When B is present in excess (B is on the “E356K:D399K side”), in all cases, the excess amount of B exists as a mixture of both homodimers and half-molecules. Even at higher ratios of 1:3 and 1:5, homodimers exist but half-molecule B is not observed. This indicates that the E356K:D399K side does not prefer homodimers as much as the KK side of the DEKK combination.

[0309] In summary, the mutation of the DEKK combination has a clearer advantage than the charge inversion CH3 mutation in that one of the chains of the heterodimer does not form a homodimer.

[0310] (Example 22: Various mixtures using the DEKK combination) The mutation of the DEKK combination has been shown to induce the formation of high-purity bispecific IgG molecules (“AB”). Next, we explored the feasibility of the controlled production of more complex antibody mixtures, such as “AB and AA” or “AB and AC” mixtures, from a single cell. The Fab types used so far were incorporated into vectors containing either the “DE construct” or the “KK construct”. And 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 based on their overall stable behavior, good expression levels, and the mass differences between the IgG containing these Fabs (see Table 19).

[0311]

Table 19

[0312]

Table 20

[0313] According to the SDS-PAGE analysis, most samples consisted mostly of full-length IgG, and in some cases, a low proportion of half-molecules was present. Furthermore, many samples showed two bands at approximately 150 kDa in the gel under non-reducing conditions. This reflects the presence of two distinguishable IgG species in the samples. Two heavy-chain bands could also be seen in some samples on the gel under reducing conditions (data not shown).

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

[0315] Although found after nMS, in sample 11, a species different from the one corresponding to the observed IgG mass was predicted. It was concluded that this was a transfection error. That is, it was revealed that in sample 11, instead of 1122-KK, 1337-KK was co-expressed with 1025-DE.

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

[0317] The summary of the results of the bispecific ELISA (OD450 values) is shown in Table 21. The gray cells indicate the species predicted for each transfection. Generally, the experimental results match the predicted results, with exceptions shown in italics or bold. In transfections 1-3, species BC (transfection #1 and #2) or AC (transfection #3), which are cells predicted to be "negative," showed a significant background signal. From previous studies, bispecific ELISA is known to suffer from high background levels. These background levels may be due to half-molecules that may be present in the sample. Note that it was confirmed from the results of the bispecific ELISA that an error occurred in transfection #11. Species AC (bold value) instead of BC was detected.

[0318]

Table 21

[0319] (Example 23: Improved mixture of two bispecific antibodies that recognize four different epitopes (AB and CD) from single cells) In Example 12, there was a hypothesis of prediction that the mixture from transfection ZA or ZB might become a problem when moving to mass production. The reason is that the knob-into-hole mutants have been reported to be unstable, and it cannot be excluded that the CH3 domain with "knob" or "hole" dimerizes with the charge-engineered CH3 domain. As shown in the above example, novel charge-pair mutants that selectively induce heterodimerization and substantially no homodimer formation were found. The polypeptide chain with a CH3 domain having these novel charge-pair mutants can be expressed in cells together with a polypeptide chain having a charge-engineered CH3 domain known so far or SEED bodies, and as a result, only two bispecific molecules can be selectively formed.

[0320] As is clear from the above example, the DEKK combinatorial mutation is excellent for producing one bispecific molecule (AB) or two bispecific molecules (AB and AC) in clonal cells where heavy-chain dimerization is induced by the CH3 domain. However, if there is only one set of vectors that can be used as complementary CH3 mutations, the types of mixtures that can be produced will be limited. If there is a second set of "orthogonal" vectors that can be used in combination with DEKK, it will be 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 "cross" dimers. "Cross" means that the heavy chain produced by one set of vectors forms a full-length IgG dimerized with the heavy chain expressed by the other set of vectors.

[0322] To test whether such "crossed" dimers can be formed, in silico analysis was performed using HADDOCK to obtain insights into whether pairing occurs between the wild-type CH3 domain and CH3 domains containing DE- or KK-mutations. Similarly, it was analyzed whether pairing can occur between the wild-type CH3 domain and CH3 domains containing E356K, D399K or K392D, K409D mutations, whether pairing can occur between the wild-type CH3 domain and CH3 domains containing knob-into-hole mutations, and for any combination of the above. The list of combinations of CH3 mutations analyzed by HADDOCK is shown in Table 22. The resulting HADDOCK scores are summarized in Figure 25.

[0323]

Table 22

[0324] As shown in Figure 25, based on these HADDOCK predictions, it seems that the combination of the CH3 of the DEKK combination and the CH3 of the charge-reversal combination is the most successful. This combination results in no by-products (especially AC, AD, BC, BD) being incorporated when co-expressed in a single cell and can form the two bispecific molecules (AB and CD) of the desired combination.

[0325] As seen in Figure 25, these undesired bispecific species AC, AD, BC and BD have relatively high HADDOCK scores. On the other hand, the desired AB and CD have the lowest HADDOCK scores. Of course, when either the DEKK or charge-reversal CH3 combination is 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 mainly produced when co-expressed in cells.

[0326] In contrast, when looking at the prediction for co-expression of DEKK and the wild type, the HADDOCK scores for AC and AD are lower than the HADDOCK score for CD. This indicates that when attempting to produce a mixture of AB and CD by co-expressing the vector encoding the CH3 combination of DEKK and the vector encoding the wild-type CH3, AC and AD are very likely to be contaminated.

[0327] Finally, in the prediction for co-expression of either DEKK or the charge-reversal mutant together with the knob-into-hole mutant, the undesirable bispecific mutants had relatively low HADDOCK scores. That is, there is a high probability that these undesirable species will be produced during co-expression.

[0328] Therefore, it was concluded that the combination of the CH3 of the DEKK combination and the CH3 of the charge-reversal combination (E356K, D399K / K392’D, K409D’) is ideally preferable for obtaining a substantially pure mixture of antibodies of “AB and CD” and / or “AB and CC”.

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

[0330] To enable the analysis of native MS, the mass difference between (predicted) species must be sufficient, i.e., greater than 190 Da. Four individual VHs were selected such that the species predicted upon co-expression would take on mass values such that they could be identified and separated by nMS. Further, the four selected VHs have a large enough mass difference to identify most of the contaminants that could arise in the mixture, in addition to the two desired species. 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 either "DE" or "KK" constructs or charge reversal constructs, and several co-expressions were performed. As before, all vectors also contain a nucleic acid encoding the common light chain IGKV1-39. As shown previously, an important requirement when combining two sets of vectors is that the heavy chains expressed from the two different sets of CH3-engineered vectors do not form "cross" dimers. By "cross" it is meant 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. To test the possibility of "cross" dimer formation between heavy chains containing charge reversal mutations and heavy chains containing DE or KK mutations, control transfections were performed.

[0333] [Table 24]

[0334] Table 25 provides further summaries of the predicted masses of the species and the contaminants that could be present in transfection numbers 9 - 11 of Table 24.

[0335] [Table 25]

[0336] Samples of all purified proteins obtained in Transfections #1-11 were analyzed by SDS-PAGE and included three control samples (Figure 26). Furthermore, nMS analysis was performed on protein samples from Transfections #9-11 to identify all species of the samples.

[0337] As seen in Figure 26, Transfection #3 and Transfection #4 resulted in a predicted 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 the amount of full-length IgG molecules.

[0338] Transfections #7 and #8 resulted in the presence of approximately equal amounts of half-molecules and full-length IgG in the protein samples. However, SDS-PAGE could not deduce which of the DE / DE dimer, DE / E356K:D399K dimer, or DE / K392D:K409D dimer the full-length IgG represented. Notably, substantially no half-molecules were observed in the samples from Transfections #9-11.

[0339] Figure 27 shows the results of the nMS analysis of Transfections #9 and #11. The predicted species ratios and contaminating species were calculated by the peak heights. In Transfection #9, the predicted species "AB and CD" represented 97% of the mixture (30% AB and 67% CD). On the other hand, only approximately 3% of contaminating BD was present (Figure 27A). In Transfection #11, the predicted species "AB and CC" represented 94% of the mixture (33% AB and 61% CC). On the other hand, only 6% of contaminating BC (4.1%) and AC (1.8%) were present (Figure 27B).

[0340] As these data show, 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 mixtures of "AB and CD" or "AB and CC". When the charge inversion construct and the DEKK construct are used in combination together, very limited amounts of "cross" dimer formation occur. By adjusting the transfection ratio, it is predicted that these by-products that are incorporated at low rates can be further reduced.

[0341] (Example 24: Single-dose pharmacokinetic study in mice) To study the pharmacokinetic (pK) behavior of bispecific antibodies retaining combinations of DEKK mutations in the CH3 region, in this study, the pK parameters of three different IgG batches were measured and compared. The three batches contained the following: 1) the parental antibody 1337:1337 of wild-type anti-tetanus toxin (two MF1337Fabs on the wild-type Fc backbone), 2) the parental antibody 1516:1516 of wild-type anti-tetanus toxin (two MF1516Fabs on the wild-type Fc backbone), 3) the CH3-engineered bispecific anti-tetanus toxin antibody 1516:1337 retaining the DEKK combination mutation in the Fc region (MF1516Fab on the DE side and MF1337Fab on the KK side).

[0342] The product of the DEKK-bispecific antibody was selected such that the parental antibodies have the specificities of 1337:1337 and 1516:1516. The reason is that it has been known from previous studies that there is no pre-formulation serum response to these antibodies in some mouse strains. Incidentally, the presence of a serum response before formulation makes it difficult to examine the research results. Furthermore, nMS also shows that there is a sufficient mass difference between the parental antibodies to enable discrimination between 1337:1337 (wild-type Fc), 1516:1337 (DEKK Fc), and 1516:1516 (wild-type Fc).

[0343] Three batches of IgG were prepared as previously described. However, the DNA used for transfection was prepared using an endotoxin-free maxiprep kit to minimize the amount of endotoxin. The batches were then tested for protein concentration, amount of aggregation, amount of endotoxin and percentage of bispecific product. It was shown to meet the acceptance criteria for use of the IgG batches in subsequent pK studies. That is, the IgG concentration after gel filtration was higher than 0.3 mg / ml, the amount of aggregation was lower than 5%, the amount of endotoxin was 3 EU / mg protein, and the DEKK batch contained more than 90% bispecific IgG.

[0344] In native mass spectrometry of the samples after gel filtration, the predicted species were present at a high percentage. In sample 1516:1337, a small amount of DE:DE homodimer, estimated to be about 2%, was detected (Figure 28). It was concluded that the three batches of IgG 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 of human IgG (5 ml / kg immunoglobulin solution / kg body weight). The animals were 7 - 8 weeks old at the time of administration and had a body weight of approximately 18 - 20 grams. Blood samples were collected before administration, 15 and 60 minutes after administration, and at 2, 4, 8, 24, 48, 96, 168, 268 and 336 hours after administration. Serum samples were prepared and stored at a temperature lower than -20 degrees until analysis. Each group consisted of three subgroups of four mice. That is, 12 mice / group. Samples were collected from each mouse at six time points.

[0346] The welfare of the mice was maintained in accordance with the European Community Directive on the Protection of Animals Used for Experimental and Other Scientific Purposes (Directive 86 / 609 / EEC) and Dutch legislation (the Experiments on Animals Act, 1997). This study was also conducted in compliance with the Standards for Humane Care and Use of Laboratory Animals, Identification Number 45859-01 (Expiration Date: April 30, 2015), issued by the Office of Laboratory Animal Welfare of the National Institutes of Health, USA.

[0347] Mice in Group 1 received an injection of the full-length monoclonal IgG antibody 1516:1516 (triangle). Mice in Group 2 received an injection of the full-length monoclonal IgG antibody 1337:1337 (square). Mice in Group 3 received an injection of the full-length bispecific IgG antibody 1516:1337 with a DEKK-engineered CH3 domain (DE side: 1516, KK side: 1337) (diamond).

[0348] The ELISA assay was used to quantitatively analyze monoclonal human antibodies in mouse sera using a quantitative human IgG ELISA kit (ZeptoMetrix, NY, USA; ELISA Kit No. 0801182). Briefly, the ELISA assay was based on the following principle. The 96-well ELISA plate was coated with anti-human IgG, and the human monoclonal antibody bound to the anti-human IgG. The bound antibody was then visualized using a horseradish peroxidase (HRP)-conjugated polyclonal anti-human IgG antibody.

[0349] The absorbance (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 bispecific IgG full-length retaining the combination of DEKK mutations was significantly similar to the parental monospecific antibody. The CH3 mutations in the DEKK-bispecific antibody do not change the stability or half-life. Also, the DEKK mutants behave like wild-type IgG.

[0350] (References) 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 Ellerson JR., et al., J. Immunol 1976 (116) 510-517; Lee and Richards J. Mol. Biol. 1971(55)379. Gunasekaran et al J.Biol.Chem. 2010(285)19637-19646 De Vries Nature Protocols 2010(5)883 Kabat et al, (1991) Mammalian Cell Biotechnology : a Practical Approach (M. Butler, ed., IRL Press, 1991 Merchant Nature biotechnology 1998(16)677 Ridgeway Protein Engineering 1996(9)617-621. Davis JH. Et al., Protein Engineering, Design & Selection 2010(23)195-202 Papadea and Check. Crit Rev Clin Lab Sci. 1989;27(1):27-58. Tissue Culture, Academic Press, Kruse and Paterson, editors (1973) Ionescu et al., J. Pharm. Sci. 2008 (97)1414) Current protocols in Protein Science 1995, coligan JE et al., Wingfield PT, ISBN 0-471-11184-8, Bendig 1988. Capelle, M.A.H., Brugger, P., Arvinte, T.Vaccine 23 (2005), 1686-1694. Demeule, B., Lawrence, M.J., Drake, A.F., Gurny, R., Arvinte, T. Biochim. Biophys. Acta 1774 (2007a), 146-153. Demeule, B., Gurny, R., Arvinte, T., Int. J. Pharm 329 (2007b), 37-45. Lakowicz, J.R., Principles of fluorescence spectroscopy; Second Edition Kluwer Academic / Plenum Publishers, New York, Boston, Dordrecht, London, Moscow, (2006) ISBN 0-306-46093-9.

Claims

1. 1. A method for producing heterodimeric IgG-like molecules from a single host cell, comprising: the IgG-like molecule 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 including giving the polypeptide chain comprising the first CH3 domain comprises an arginine (R) residue at amino acid position 366, according to the EU numbering system; the polypeptide chain comprising the second CH3 domain comprises an aspartic acid (D) or glutamic acid (E) residue at amino acid position 351, according to the EU numbering system; The method further comprises culturing the host cell to express the two nucleic acid molecules and recovering the heterodimeric IgG-like molecule 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. 3. The method according to claim 1 or 2, wherein the polypeptide chain comprising the first CH3 domain further comprises a lysine (K) residue at amino acid position 351, according to the EU numbering system.

4. 4. The method according to claim 1, wherein wherein the polypeptide chain comprising the second CH3 domain further comprises a glutamic acid (E) or aspartic acid (D) residue at amino acid position 349 according to the European Union numbering system, and / or a glutamic acid (E) residue at amino acid position 368 according to the European Union numbering system.

5. 5. The method of claim 4, wherein the polypeptide chain comprising the second CH3 domain further comprises a glutamic acid (E) residue at amino acid position 368, according to the EU numbering system.

6. 6. The method according to any one of claims 1 to 5, The presence of contaminating homodimers is less than 5%.

7. 7. The method of claim 6, The presence of contaminating homodimers is less than 2%.

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

9. 9. The method of claim 8, A method in which each of the two variable regions of the polypeptide chain comprising the CH3 domain recognizes a different target epitope.

10. 10. The method of claim 9, The method, wherein said different target epitopes are located on the same target molecule.

11. 11. The method of claim 10, The method, wherein the target molecule is a water-soluble molecule.

12. 11. The method of claim 10, The method, wherein the target molecule is a membrane-bound molecule.

13. 10. The method of claim 9, The method, wherein said different target epitopes are located on different target molecules.

14. 14. The method of claim 13, The different target molecules are expressed in the same cell.

15. 14. The method of claim 13, The method, wherein the different target molecules are expressed in different cells.

16. 14. The method of claim 13, The method, wherein the different target molecules are water-soluble molecules.

17. 14. The method of claim 13, A method wherein one target molecule is a water-soluble molecule and the second target molecule is a membrane-bound molecule.

18. 18. The method of any one of claims 8 to 10, 12 to 15, or 17, The method, wherein at least one of the target epitopes is located on a tumor cell.

19. 18. The method of any one of claims 8 to 10, 12 to 15, or 17, The method, wherein at least one of said target epitopes is located on an effector cell.

20. 20. The method of claim 19, 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.

21. 21. The method of claim 19 or 20, The method, wherein the target epitope is located on a CD3, CD16, CD25, CD28, CD64, CD89, NKG2D, or NKp46 molecule.

22. 22. The method of any one of claims 1 to 21, The method, wherein said heterodimeric IgG-like molecule is an antibody.

23. 23. The method of any one of claims 1 to 22, The method, wherein said heterodimeric IgG-like molecule is a human IgG.

24. 24. The method of any one of claims 1 to 23, The method, wherein said heterodimeric IgG-like molecule is human IgG1.

25. 25. The method of any one of claims 2 to 24, The method, wherein the common light chain is a germline-derived light chain.

26. 26. The method of any one of claims 2 to 25, The method, wherein the common light chain is a rearranged germline human kappa light chain IgVκ1-39*01 / IGJκ1*01.

27. 27. The method of any one of claims 1 to 26, 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; 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.

28. 28. A heterodimeric IgG-like molecule obtainable by the method of any one of claims 1 to 27.

29. 29. The heterodimeric IgG-like molecule of claim 28, The heterodimeric IgG-like molecules bind to different epitopes on the same antigen and / or different epitopes on different antigens.

30. 30. The heterodimeric IgG-like molecule of claim 28 or 29, A heterodimeric IgG-like molecule, wherein said IgG-like molecule is an antibody.

31. A heterodimeric antibody comprising two CH3 domains, one of the two CH3 domains comprises an arginine (R) residue at amino acid position 366, according to the EU numbering system; A heterodimeric antibody, wherein the other of the two CH3 domains comprises an aspartic acid (D) or glutamic acid (E) residue at amino acid position 351 according to the EU numbering system.

32. 1. A recombinant host cell comprising a nucleic acid sequence encoding a polypeptide chain comprising at least a first and a second CH3 domain, the polypeptide chain comprising the first CH3 domain comprises an arginine (R) residue at amino acid position 366, according to the EU numbering system; A recombinant host cell, wherein the polypeptide chain comprising the second CH3 domain comprises an aspartic acid (D) or glutamic acid (E) residue at amino acid position 351, according to the EU numbering system.

33. 33. The recombinant host cell of claim 32, A recombinant host cell, wherein the host cell further comprises a nucleic acid sequence encoding a common light chain.

34. A pharmaceutical composition comprising the heterodimeric IgG-like molecule of any one of claims 28 to 30 or the heterodimeric antibody of claim 31 and a pharmaceutically acceptable carrier.

35. 35. The pharmaceutical composition of claim 34, A pharmaceutical composition, wherein the heterodimeric IgG-like molecule or the heterodimeric antibody is produced in a recombinant host cell according to claim 32 or claim 33.

36. 1. A method of producing a host cell for producing a heterodimeric IgG-like molecule, comprising: The method comprises: introducing into the host cell a nucleic acid sequence encoding a polypeptide chain comprising at least a first and a second CH3 domain; the polypeptide chain comprising the first CH3 domain comprises an arginine (R) residue at amino acid position 366, according to the EU numbering system; the polypeptide chain comprising the second CH3 domain comprises an aspartic acid (D) or glutamic acid (E) residue at amino acid position 351, according to the EU numbering system; The method wherein said nucleic acid sequences are introduced sequentially or simultaneously.

37. 37. The method of claim 36, The method further comprising introducing into said host cell a nucleic acid sequence encoding a common light chain.

38. 38. The method of claim 36 or 37, 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; 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.

39. 39. A culture of a recombinant host cell according to claim 32 or 33, or of a recombinant host cell obtained by the method of any one of claims 36 to 38, which produces a heterodimeric IgG-like molecule.

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