METHOD FOR PRODUCING AND PURIFYING A POLYPEPTIDIC MULTIMER

专利摘要:
method for producing a polypeptide multimer, polypeptide multimers, method for purifying a polypeptide multimer, nucleic acid, vector, cell, and pharmaceutical composition. the present invention provides efficient methods based on altering the binding affinity to protein a, to produce or purify multispecific antibodies that have the activity of binding two or more types of antigens with high purity by only one purification step based on a protein. the methods of the present invention for producing or purifying multispecific antibodies that characterize them with altered amino acid residues of the constant region and / or variable region of the antibody heavy chain. multispecific antibodies with altered protein-a binding activity, which exhibit plasma retention comparable or longer than that of human igg1, can be efficiently prepared in high purity by introducing amino acid changes of the present invention into antibodies.
公开号:BR112012017124B1
申请号:R112012017124-0
申请日:2010-12-24
公开日:2021-04-06
发明作者:Tomoyuki Igawa；Zenjiro Sampei；Tetsuya Wakabayashi；Eriko Ito
申请人:Chugai Seiyaku Kabushiki Kaisha；
IPC主号:

专利说明:

[0001] [0001] The present invention relates to methods for producing or purifying polypeptide multimers, polypeptide multimers with an altered protein A binding capacity, and the like. Reference Technique
[0002] [0002] There are some previously reported methods for producing bispecific IgG-like antibodies having a human constant region (IgG-like antibody having a human constant region and in which one arm has a specific activity for antigen A and the other has an activity specific binding to antigen B). In general, bispecific IgG-type antibody is composed of two types of H chains (ie, H chain against antigen A and H chain against antigen B) and two types of L chains (ie, chain L against antigen A and L chain against B antigen). When such a bispecific IgG-like antibody is expressed, two types of H chains and two types of L chains are expressed, and there are ten possible combinations of the H2L2 combination. Of these, only one combination has the specificity of interest (one arm has specific binding activity to antigen A and the other has specific binding activity to antigen B). Thus, in order to obtain a bispecific antibody of interest, it is necessary to purify a single antibody of interest from ten types of antibodies. This is an extremely inefficient and difficult process.
[0003] [0003] There are reported methods for solving this problem that use a common L chain so that the L chain against antigen A and the L chain against antigen B have an identical amino acid sequence (Patent Documents 1 and 2). When bispecific IgG type antibodies having such a common L chain are expressed, two types of H chains and one type of common L chain are expressed, and there are three possible combinations for the H2L2 combination. One of these combinations is a bispecific antibody of interest. These three combinations are: monospecific antibody against antigen A (homomeric H chain antibody against antigen A), bispecific antibody against both antigen A and against antigen B (heteromeric antibody with an H chain against antigen A and a H chain against antigen B), and monospecific antibody against B antigen (homomeric H chain antibody against B antigen). Since its proportion is generally 1: 2: 1, the expression efficiency of the desired bispecific antibody is approximately 50%. A method of further improving this efficiency has been reported that allows two types of heteromerically associated H chains (Patent Document 3). This can increase the expression efficiency of the desired bispecific antibody up to approximately 90-95%. However, a method has been reported to efficiently remove two types of homomeric antibodies that are impurities, in which amino acid substitutions are introduced in the variable regions of two types of H chains to provide them with different isoelectric points so that two types of homomeric antibodies and the bispecific antibody of interest (heteromeric antibody) can be purified by ion exchange chromatography (Patent Document 4). A combination of the aforementioned methods allowed to efficiently produce a bispecific antibody (heteromeric antibody) having an IgG-like human constant region.
[0004] [0004] On the other hand, in industrial production of IgG-type antibodies, a purification step by chromatography on protein A should be used, but ion exchange chromatography is not necessarily used in the purification step. Therefore, the use of ion exchange chromatography to produce a highly pure bispecific antibody leads to increased production costs. In addition, since ion exchange chromatography alone cannot ensure a robust method of purifying pharmaceutical products, it is preferable to perform more than one chromatographic step to remove impurities.
[0005] [0005] In any case, it is preferable that bispecific antibodies can also be highly purified by a chromatographic step that has a different mode of separation than that of ion exchange chromatography. It is desirable that as one of such modes of separation, protein A chromatography, which is to be used in the industrial production of IgG type antibodies, is capable of purifying bispecific antibodies at high purity.
[0006] [0006] A previously reported method for purifying a bispecific antibody (heteromeric antibody) using protein A is to use a bispecific antibody having a mouse IgG2a H chain that binds to protein A and a non-rat IgG2b H chain. binds to protein A. It has been reported that this method allows a bispecific antibody of interest to be purified to 95% purity by the protein A-based purification step alone (Non-Patent Document 1 and Patent Document 5). However, this method also uses ion exchange chromatography to improve the purity of the bispecific antibody. In other words, purification of a highly pure bispecific antibody cannot be achieved by the purification step using only protein A chromatography. In addition, catumaxomab, a bispecific antibody produced by the method described above and having a mouse IgG2a H chain and a mouse IgG2b H chain, has a half-life of approximately 2.1 days in humans, which is extremely shorter than that of normal human IgG1 (2 to 3 weeks) (Non-Patent Document 2). In addition to having a short half-life, catumaxomab is highly immunogenic because of its constant regions of mice and rats (Non-Patent Document 3). Thus, a bispecific antibody obtained by such methods is considered inappropriate as a pharmaceutical product.
[0007] [0007] On the other hand, it has been suggested that from the point of view of immunogenicity, a human IgG3 constant region could be used as a constant region of non-binding to protein A (Non-Patent Document 1). However, as it is known that the human IgG1 and human IgG3 H chains only associate with each other (Non-Patent Document 1), it is impossible to produce a bispecific antibody of interest using a human IgG1 H chain and a human IgG3 H chain by same method used for the bispecific antibody having a mouse IgG2a H chain and a mouse IgG2b H chain. In addition, it has been reported that the half-life of human IgG3 in humans is generally shorter than that of human IgG1, human IgG2, and human IgG4 (Non-Patent Documents 4 and 5). Consequently, like the bispecific antibody using a mouse IgG2a and a rat IgG2b, a bispecific antibody using human IgG3 could also have a short half-life in human. It is suggested that the reason that the H chain association rarely occurs between human IgG1 and human IgG3 is the hinge sequence of human IgG3 (Non-Patent Document 1). However, the reason for the short half-life of the human IgG3 constant region has not been fully elucidated yet. Thus, there have been no reports for the moment regarding bispecific antibodies that use a human IgG3 constant region as a constant region of non-binding to protein A. Furthermore, there are also no reports as to methods for the efficient production or purification of bispecific antibodies. highly pure that have a human constant region and show a similarly long half-life as human IgG1. Prior Art Documents Patent Documents Patent Document 1: WO98050431 Patent Document 2: WO2006109592 Patent Document 3: WO2006106905 Patent Document 4: WO2007114325 Patent Document 5: WO95033844 Non-Patent Documents Non-Patent Document 1: The Journal of Immunology, 1995, 155: 219-225 Non-Patent Document 2: J Clin Oncol 26: 2008 (May 20 suppl; abstr 14006) Non-Patent Document 3: Clin Cancer Res 2007 13: 38993905 Non-Patent Document 4: Nat Biotechnol. 2007 Dec; 25 (12): 1369-72 Non-Patent Document 5: J. Clin Invest 1970; 49: 673-80 Disclosure of the Invention [Problems to be solved by the invention]
[0008] [0008] In general, an ordinary IgG-like antibody can be efficiently produced as a highly pure IgG by a protein A-based purification step. However, the production of a highly pure bispecific antibody requires an additional purification step using exchange chromatography. ionic. The addition of such a purification step by ion exchange chromatography can complicate the production and increase the cost of production. Thus, it is preferable to produce a highly pure bispecific antibody by a purification step based on protein A alone. An object of the present invention is to provide methods that use only one protein A-based purification step to efficiently produce or purify highly pure IgG-like bispecific antibody having a constant region of the human antibody heavy chain.
[0009] [0009] However, since the protein A binding site in the Fc domain is identical to the FcRn binding site in the Fc domain, it is expected to be difficult to adjust protein A binding activity while conserving the human FcRn binding . Conserving the ability to bind to human FcRn is very important for long plasma retention (long half-life) in humans that is characteristic of IgG-like antibodies. The present invention provides methods that use only one protein A-based purification step to efficiently produce or purify a highly pure bispecific antibody that maintains a plasma retention time comparable to or longer than that of human IgG1. [Means to Solve Problems]
[0010] [00010] The present inventors have discovered methods that use only one protein A-based purification step to efficiently purify or produce a highly pure polypeptide multimer capable of binding to two or more antigens, especially a multispecific IgG type antibody having a human constant region , altering its ability to bind to protein A.
[0011] [00011] Furthermore, these methods have been combined with methods to regulate the association between a first polypeptide having an antigen binding activity and a second polypeptide having an antigen binding activity by modifying amino acids that form the interface formed in the association of the polypeptides. By this combination, the present invention allows for the efficient production or purification of a highly pure polypeptide multimer of interest.
[0012] [00012] The present inventors also found that by modifying the amino acid residue at position 435 (EU numbering) in the heavy chain constant region, the protein A binding capacity can be adjusted while maintaining its plasma retention comparable or longer than that of Human IgG1. Based on this finding, a highly pure bispecific antibody with a plasma retention time comparable or longer than that of human IgG1 can be produced or purified.
[0013] [00013] The present invention is based on the findings described above, and provides [1] to [55] below:
[0014] (a) expressão de um DNA que codifica o primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e um DNA que codifica o segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou sem atividade de ligação ao antígeno; e (b) coleta do produto de expressão da etapa (a), em que um ou mais resíduos de aminoácido em um ou tanto no primeiro polipeptídeo tendo uma atividade de ligação ao antígeno quanto no segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou sem atividade de ligação ao antígeno foram modificados, para que haja uma maior diferença da capacidade de ligação à proteína A entre o primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e o segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou sem atividade de ligação ao antígeno.[00014] [1] A method for producing a polypeptide multimer comprising a first polypeptide having antigen binding activity and a second polypeptide having antigen binding activity or without antigen binding activity, comprising the steps of : (a) expression of a DNA encoding the first polypeptide having antigen binding activity and a DNA encoding the second polypeptide having antigen binding activity or without antigen binding activity; and (b) collecting the expression product from step (a), where one or more amino acid residues in one or both of the first polypeptide having antigen-binding activity and the second polypeptide having antigen-binding activity or without antigen-binding activity have been modified, so that there is a greater difference in protein A binding capacity between the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or without antigen binding activity.
[0015] [00015] [2] The method of [1], in which the expression product is collected using protein A affinity chromatography in step (b).
[0016] [00016] [3] The method of [1] or [2], in which one or more amino acid residues in one or both of the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or without antigen binding activity have been modified, so that there is a greater difference between the solvent pH to elute the first polypeptide having a protein A antigen binding activity and that to elute the second polypeptide having antigen binding activity or without protein A antigen-binding activity
[0017] [00017] [4] The method of any one of [1] to [3], wherein one or more amino acid residues in the first polypeptide having an antigen binding activity or in the second polypeptide having an antigen binding activity or without antigen binding activity were modified to increase or reduce the protein A binding capacity of one of the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or no antigen binding activity antigen.
[0018] [00018] [5] The method of any one from [1] to [4], wherein one or more amino acid residues in the first polypeptide having an antigen binding activity and in the second polypeptide having an antigen binding activity or without antigen binding activity were modified to increase protein A binding capacity of one of the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or no antigen binding activity, and reducing the protein A binding capacity of another polypeptide.
[0019] [00019] [6] The method of any one from [1] to [5], in which the purity of the collected polypeptide multimer is 95% or more.
[0020] [00020] [7] The method of any of [1] to [6], wherein the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or without antigen binding activity comprises an amino acid sequence of an antibody Fc domain or an amino acid sequence of the antibody heavy chain constant region.
[0021] [00021] [8] The method of [7], in which at least one amino acid residue selected from amino acid residues of positions 250 to 255, 308 to 317, and 430 to 436 (EU numbering) in the amino acid sequence of the antibody Fc domain or constant region of the antibody heavy chain has been modified.
[0022] [00022] [9] The method of any one from [1] to [8], wherein the first polypeptide having an antigen binding activity and the second polypeptide having an antigen binding activity comprises an amino acid sequence of one variable region of the antibody heavy chain.
[0023] [00023] [10] The method of [9], wherein at least one amino acid residue has been modified in the amino acid sequences of FR1, CDR2, and FR3 of the variable region of the antibody heavy chain.
[0024] [00024] [11] The method of any one from [1] to [10], wherein the polypeptide multimer comprises one or two third polypeptides having an antigen binding activity, and step (a) comprises the expression of a DNA which encodes the third polypeptide having antigen-binding activity.
[0025] [00025] [12] The method of [11], wherein the third polypeptide having antigen binding activity comprises an amino acid sequence of an antibody light chain.
[0026] [00026] [13] The method of [11] or [12], wherein the polypeptide multimer additionally comprises a fourth polypeptide having antigen binding activity, and step (a) comprises the expression of a DNA encoding the fourth polypeptide having antigen-binding activity.
[0027] [00027] [14] The method of [13], wherein at least one of the third and fourth polypeptides having an antigen binding activity comprises an amino acid sequence of an antibody light chain.
[0028] [00028] [15] The method of [13], wherein the first polypeptide having an antigen binding activity comprises amino acid sequences from a variable region of the antibody light chain and the constant region of the antibody heavy chain; the second polypeptide having antigen-binding activity comprises an amino acid sequence of an antibody heavy chain; the third polypeptide having antigen-binding activity comprises amino acid sequences from a variable region of the antibody heavy chain and a constant region from the antibody light chain; and the fourth polypeptide having antigen-binding activity comprises an amino acid sequence of an antibody light chain.
[0029] [00029] [16] The method of any one from [1] to [15], wherein the polypeptide multimer is a multispecific antibody.
[0030] [00030] [17] The method of [16], wherein the multispecific antibody is a bispecific antibody.
[0031] [00031] [18] The method of any one from [1] to [8], which comprises the first polypeptide having an antigen binding activity and the second polypeptide having no antigen binding activity, and wherein the first polypeptide having an antigen binding activity comprises an amino acid sequence of an antigen binding domain of a receptor and an amino acid sequence of an antibody Fc domain, and the second polypeptide having no antigen binding activity comprises a sequence of amino acids from an antibody Fc domain.
[0032] [00032] [19] The method of any of [7] to [18], wherein the antibody Fc domain or antibody heavy chain constant region is derived from human IgG.
[0033] [00033] [20] A polypeptide multimer produced by the method of anyone from [1] to [19].
[0034] (a) expressão de um DNA que codifica o primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e um DNA que codifica o segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou sem atividade de ligação ao antígeno; e (b) coleta do produto de expressão da etapa (a) por cromatografia de afinidade em proteína A, em que um ou mais resíduos de aminoácidos em um ou tanto no primeiro polipeptídeo tendo uma atividade de ligação ao antígeno quanto no segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou sem atividade de ligação ao antígeno foram modificados, para que haja uma maior diferença da capacidade de ligação à proteína A entre o primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e o segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou sem atividade de ligação ao antígeno.[00034] [21] A method for purifying a polypeptide multimer comprising a first polypeptide having antigen binding activity and a second polypeptide having antigen binding activity or without antigen binding activity, comprising the steps of : (a) expression of a DNA encoding the first polypeptide having antigen binding activity and a DNA encoding the second polypeptide having antigen binding activity or without antigen binding activity; and (b) collecting the expression product from step (a) by protein A affinity chromatography, where one or more amino acid residues in one or both of the first polypeptide having antigen-binding activity and the second polypeptide having antigen-binding activity or without antigen-binding activity have been modified, so that there is a greater difference in protein A binding capacity between the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or without antigen binding activity.
[0035] [00035] [22] The method of [21], in which one or more amino acid residues in the first polypeptide having antigen binding activity or the second polypeptide having antigen binding activity or without antigen binding activity were modified, to increase or reduce the protein A binding capacity of the first polypeptide having antigen binding activity or the second polypeptide having antigen binding activity or without antigen binding activity.
[0036] [00036] [23] The method of [20] or [21], in which one or more amino acid residues in the first polypeptide having antigen binding activity and in the second polypeptide having antigen binding activity or without antigen activity antigen binding were modified to increase the protein A binding capacity of one of the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or no antigen binding activity, and reducing the ability to bind to protein A from another polypeptide.
[0037] [00037] [24] The method of any one from [21] to [23], in which the purity of the collected polypeptide multimer is 95% or more.
[0038] [00038] [25] The method of any one from [21] to [24], wherein the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or without antigen binding activity comprises an amino acid sequence of an antibody Fc domain or an amino acid sequence of the antibody heavy chain constant region.
[0039] [00039] [26] The method of [25], in which at least one amino acid residue selected from amino acid residues of positions 250 to 255, 308 to 317, and 430 to 436 (EU numbering) in the amino acid sequence of the antibody Fc domain or constant region of the antibody heavy chain has been modified.
[0040] [00040] [27] The method of any one of [21] to [26], wherein the first polypeptide having an antigen binding activity and the second polypeptide having an antigen binding activity comprises an amino acid sequence of one variable region of the antibody heavy chain.
[0041] [00041] [28] The method of [27], wherein at least one amino acid residue has been modified in the amino acid sequences of FR1, CDR2, and FR3 of the variable region of the antibody heavy chain.
[0042] [00042] [29] The method of any one of [21] to [28], wherein the polypeptide multimer comprises one or two third polypeptides having an antigen binding activity, and step (a) comprises the expression of a DNA which encodes the third polypeptide having antigen-binding activity.
[0043] [00043] [30] The method of [29], wherein the third polypeptide having antigen binding activity comprises an amino acid sequence of an antibody light chain.
[0044] [00044] [31] The method of [29] or [30], wherein the polypeptide multimer additionally comprises a fourth polypeptide having antigen binding activity, and step (a) comprises the expression of a DNA encoding the fourth polypeptide having antigen-binding activity.
[0045] [00045] [32] The method of [31], wherein at least one of the third and fourth polypeptides having an antigen binding activity comprises an amino acid sequence of an antibody light chain.
[0046] [00046] [33] The method of [31], wherein the first polypeptide having an antigen binding activity comprises amino acid sequences of a variable region of the antibody light chain and constant region of the antibody heavy chain; the second polypeptide having antigen-binding activity comprises an amino acid sequence of an antibody heavy chain; the third polypeptide having antigen-binding activity comprises amino acid sequences from a variable region of the antibody heavy chain and a constant region from the antibody light chain; and the fourth polypeptide having antigen-binding activity comprises an amino acid sequence of an antibody light chain.
[0047] [00047] [34] The method of any one from [21] to [33], wherein the polypeptide multimer is a multispecific antibody.
[0048] [00048] [35] The method of [34], wherein the multispecific antibody is a bispecific antibody.
[0049] [00049] [36] The method of any one from [25] to [35], wherein the antibody Fc domain or antibody heavy chain constant region is derived from human IgG.
[0050] [00050] [37] A polypeptide multimer comprising a first polypeptide having antigen binding activity and a second polypeptide having antigen binding activity or without antigen binding activity, wherein protein A binding capacity is different for the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or without antigen binding activity.
[0051] [00051] [38] The polypeptide multimer of [37], in which there is a difference between the solvent pH to elute the first polypeptide having a binding activity to the protein A antigen and that to elute the second polypeptide having a binding activity antigen or without antigen-binding activity of protein A.
[0052] [00052] [39] The polypeptide multimer of [37] or [38], wherein the first polypeptide having antigen binding activity or the second polypeptide having antigen binding activity or without antigen binding activity comprises a amino acid sequence of an antibody Fc domain or an amino acid sequence of the antibody heavy chain constant region, and wherein at least one amino acid residue selected from the amino acid residues of positions 250 to 255, 308 to 317, and 430 to 436 (EU numbering) in the amino acid sequence of the antibody Fc domain or constant region of the antibody heavy chain has been modified.
[0053] [00053] [40] The polypeptide multimer of any one from [37] to [39], wherein the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or without antigen binding activity antigen comprises an amino acid sequence of an antibody Fc domain or an amino acid sequence of the antibody heavy chain constant region; wherein the amino acid residue of position 435 (EU numbering) in the amino acid sequence of the antibody Fc domain or the antibody heavy chain constant region is histidine or arginine in one of the first polypeptide having an antigen binding activity and the second polypeptide having antigen-binding activity or without antigen-binding activity; and wherein the amino acid residue of position 435 (EU numbering) in the amino acid sequence of the antibody Fc domain or the antibody heavy chain constant region in any of said polypeptides is different from that in another polypeptide.
[0054] [00054] [41] The polypeptide multimer of any one from [37] to [40], wherein the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or without antigen binding activity antigen comprises an amino acid sequence of an antibody Fc domain or an amino acid sequence of the antibody heavy chain constant region; wherein the amino acid residue of position 435 (EU numbering) in the amino acid sequence of the antibody Fc domain or constant region of the antibody heavy chain is histidine in one of the first polypeptide having an antigen binding activity and the second polypeptide having a antigen-binding activity or without antigen-binding activity; and wherein the amino acid residue of position 435 (EU numbering) in the amino acid sequence of the antibody Fc domain or constant region of the antibody heavy chain is arginine in another polypeptide.
[0055] [00055] [42] The polypeptide multimer of any one from [37] to [41], wherein the first polypeptide having an antigen binding activity and the second polypeptide having an antigen binding activity comprises an amino acid sequence of a variable region of the antibody heavy chain, and at least one amino acid residue was modified in the amino acid sequences of FR1, CDR2, and FR3 of the variable region of the heavy chain.
[0056] [00056] [43] The polypeptide multimer of any one from [37] to [42], which additionally comprises one or two third polypeptides having antigen-binding activity.
[0057] [00057] [44] The polypeptide multimer of [43], wherein the third polypeptide having antigen binding activity comprises an amino acid sequence of an antibody light chain.
[0058] [00058] [45] The polypeptide multimer of [43] or [44], which additionally comprises a fourth polypeptide having antigen-binding activity.
[0059] [00059] [46] The polypeptide multimer of [45], wherein at least one of the third and fourth polypeptides having antigen binding activity comprises an amino acid sequence of an antibody light chain.
[0060] [00060] [47] The polypeptide multimer of [45], wherein the first polypeptide having an antigen-binding activity comprises amino acid sequences of a variable region of the antibody light chain and constant region of the antibody heavy chain; the second polypeptide having antigen-binding activity comprises an amino acid sequence of an antibody heavy chain; the third polypeptide having antigen-binding activity comprises amino acid sequences from a variable region of the antibody heavy chain and a constant region from the antibody light chain; and the fourth polypeptide having antigen-binding activity comprises an amino acid sequence of an antibody light chain.
[0061] [00061] [48] The polypeptide multimer of any one from [37] to [47], which is a multispecific antibody.
[0062] [00062] [49] The polypeptide multimer of [48], wherein the multispecific antibody is a bispecific antibody.
[0063] [00063] [50] The polypeptide multimer of any one of [37] to [41], which comprises the first polypeptide having antigen binding activity and the second polypeptide having no antigen binding activity, and wherein the the first polypeptide having an antigen binding activity comprises an amino acid sequence of an antigen binding domain of a receptor and an amino acid sequence of an antibody Fc domain, and the second polypeptide having no antigen binding activity comprises an amino acid sequence of an antibody Fc domain.
[0064] [00064] [51] The polypeptide multimer of any one of [39] to [50], wherein the antibody Fc domain or the antibody heavy chain constant region is derived from human IgG.
[0065] [00065] [52] A nucleic acid that encodes a polypeptide that constitutes the polypeptide multimer of any one of [20] and [37] to [51].
[0066] [00066] [53] A vector inserted with the nucleic acid of [52].
[0067] [00067] [54] A cell comprising the nucleic acid of [52] or the vector [of 53].
[0068] [00068] [55] A pharmaceutical composition comprising the polypeptide multimer of any one of [20] and [37] to [51] as an active ingredient. [Effects of the Invention]
[0069] [00069] The present invention provides methods that use only one purification step based on protein A to efficiently purify or produce a highly pure polypeptide multimer having binding activity against two or more antigens (multispecific antibody), changing its protein binding capacity A. The methods of the present invention allow for purification or efficient production of a highly pure polypeptide multimer of interest without impairing the effects of other amino acid modifications of interest. Especially, by combining these methods with a method to regulate the association between two protein domains, the polypeptide multimers of interest can be more efficiently produced or purified to the highest purity.
[0070] [00070] The methods of the present invention for producing or purifying multispecific antibodies are characterized in that the amino acid residues in their constant region of the antibody heavy chain and / or variable region of the antibody heavy chain are modified. The amino acid modifications of the present invention are introduced in these regions to modify their binding capacity to protein A. In addition, other effects of the amino acid modification of interest, for example, plasma retention time comparable or longer than that of IgG1 can also be obtained. The methods of the present invention allow for the efficient preparation of highly pure multispecific antibodies having such amino acid modifying effects.
[0071] [00071] In general, the production of highly pure multispecific IgG antibodies requires a purification step using ion exchange chromatography. However, the addition of this purification step complicates the production and increases the cost of production. On the other hand, purification using only ion exchange chromatography may not be quite robust as a method of purifying pharmaceutical products. Thus, it is a task to develop a method for producing the bispecific IgG type antibody using only a protein A-based purification step, or to develop a robust production method using a protein A-based purification step and a chromatography step of ion exchange. Brief Description of Drawings
[0072] [00072] Fig. 1 is a graph showing an assessment of the plasma retention time of MRA-IgG1 and MRA-z106 / z107k in transgenic mice with human FcRn.
[0073] [00073] Fig. 2 is a diagram showing that the same region in the antibody Fc domain binds to protein A and FcRn.
[0074] [00074] Fig. 3 shows a time course of plasma concentrations of Q499-z118 / J339-z119 / L377-k and Q499-z121 / J339-z119 / L377-k after administration to transgenic mice with human FcRn.
[0075] [00075] Fig. 4 is a schematic diagram of a GC33-IgG1-CD3-scFv molecule that divally binds to the specific cancer glypican-3 antigen (GPC3) and monovalently binds to the CD3 T cell antigen.
[0076] [00076] Fig. 5 shows the result of analysis of NTA1L / NTA1R / GC33-k0 and NTA2L / NTA2R / GC33-k0 purified with protein size exclusion chromatography A.
[0077] [00077] Fig. 6 is a schematic diagram of an anti-GPC3 IgG antibody molecule that monovalently binds to glypican-3.
[0078] [00078] Fig. 7 shows the result of analysis of NTA4L-cont / NTA4R-cont / GC33-k0, NTA4L-G3 / NTA4R-cont / GC33-k0, and NTA4L / NTA4R / GC33-k0 purified with exclusion chromatography of protein size A.
[0079] [00079] Fig. 8 shows chromatograms of NTA4L-cont / NTA4R-cont / GC33-k0, NTA4L-G3 / NTA4R-cont / GC33-k0, and NTA4L / NTA4R / GC33-k0 subjected to purification with column chromatography protein A with pH gradient elution.
[0080] [00080] Fig. 9 is a schematic diagram of an alpha Fc-Fc receptor fusion protein molecule that monovalently binds to IgA.
[0081] [00081] Fig. 10 shows the result of analysis of IAL-cont / IAR-cont and IAL / IAR purified with protein size exclusion chromatography.
[0082] [00082] Fig. 11 is a schematic diagram of no1, a bispecific naturally occurring anti-IL-6 receptor / anti-GPC3 antibody.
[0083] [00083] Fig. 12 is a schematic diagram of no2, which was obtained by exchanging the VH domain and the VL domain of the anti-GPC3 antibody in no1.
[0084] [00084] Fig. 13 is a schematic diagram of no3, which was obtained by modifying no2 to change the isoelectric point of each chain.
[0085] [00085] Fig. 14 is a schematic diagram of no5, which was obtained by modifying no3 to increase the heteromeric association of H chains and to heteromerically purify associated antibody using protein A.
[0086] [00086] Fig. 15 is a schematic diagram of no6, which was obtained by modifying no5 to increase the association between the H chain of interest and the L chain of interest.
[0087] [00087] Fig. 16 are bispecific antibody chromatograms no1, no2, no3, no5 and no6 of anti-IL-6 / anti-GPC3 receptor in cation exchange chromatography to evaluate their expression models.
[0088] [00088] Fig. 17 is a CM no6 chromatogram eluted with a pH gradient from a HiTrap HP protein A column (GE Healthcare).
[0089] [00089] Fig. 18 is a chromatogram of the cation exchange chromatography analysis to assess a fraction of the main peak obtained by purifying a purified no6 fraction in protein A using an SP Sepharose HP column (GE Healthcare). Mode for Carrying Out the Invention
[0090] (a) expressão de um DNA que codifica um primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e DNA que codifica um segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou sem atividade de ligação ao antígeno; e (b) coleta dos produtos de expressão da etapa (a); em que um ou mais resíduos de aminoácidos em um ou tanto no primeiro polipeptídeo tendo uma atividade de ligação ao antígeno quanto no segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou sem atividade de ligação ao antígeno foram modificados para que haja uma maior diferença da capacidade de ligação à proteína A entre o primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e o segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou sem atividade de ligação ao antígeno.[00090] The present invention provides methods for producing a polypeptide multimer comprising a first polypeptide having antigen binding activity and a second polypeptide having antigen binding activity or without antigen binding activity. The methods of the present invention for producing a polypeptide multimer comprise the steps of: (a) expression of a DNA encoding a first polypeptide having antigen binding activity and DNA encoding a second polypeptide having antigen binding activity or without antigen binding activity; and (b) collecting the expression products from step (a); on what one or more amino acid residues in one or both of the first polypeptide having antigen-binding activity and the second polypeptide having antigen-binding activity or without antigen-binding activity have been modified so that there is a greater difference in binding capacity to protein A between the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or without antigen binding activity.
[0091] [00091] The methods of the present invention for producing a polypeptide multimer can also be expressed as methods for producing a polypeptide multimer with an altered protein A binding capacity.
[0092] [00092] In the present invention, "a polypeptide having a first antigen binding activity" can be referred to as "a first polypeptide having an antigen binding activity". "A polypeptide having a second antigen binding activity or without antigen binding activity" can be referred to as "a second polypeptide having antigen binding activity or without antigen binding activity". The same applies to "a polypeptide having a third antigen-binding activity" and "a polypeptide having a fourth antigen-binding activity" described below.
[0093] [00093] In the present invention, the term "understand" means both "understand" and "consist of".
[0094] (a) expressão de um DNA que codifica um primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e DNA que codifica um segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou sem atividade de ligação ao antígeno; e (b) coleta dos produtos de expressão da etapa (a) por cromatografia de afinidade em proteína A; em que um ou mais resíduos de aminoácidos em um ou tanto no primeiro polipeptídeo tendo uma atividade de ligação ao antígeno quanto no segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou sem atividade de ligação ao antígeno foram modificados para que a capacidade de ligação à proteína A seja diferente entre o primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e o segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou sem atividade de ligação ao antígeno.[00094] The present invention also provides methods for purifying a polypeptide multimer comprising a first polypeptide having antigen binding activity and a second polypeptide having antigen binding activity or without antigen binding activity. The methods of the present invention for purifying a polypeptide multimer comprise the steps of: (a) expression of a DNA encoding a first polypeptide having antigen binding activity and DNA encoding a second polypeptide having antigen binding activity or without antigen binding activity; and (b) collecting the expression products from step (a) by protein A affinity chromatography; on what one or more amino acid residues in one or both of the first polypeptide having antigen-binding activity and the second polypeptide having antigen-binding activity or without antigen-binding activity have been modified so that protein A-binding capacity is different between the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or without antigen binding activity.
[0095] [00095] A polypeptide having antigen-binding activity in which one or more amino acid residues have been modified can be obtained by: preparation of a DNA encoding a polypeptide having antigen-binding activity or without antigen-binding activity, modification of one or more nucleotides in DNA; introduction of the resulting DNA into cells known to those skilled in the art; cell culture to express DNA; and expression product collection.
[0096] (a) fornecimento de DNA que codifica um primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e DNA que codifica um segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou sem atividade de ligação ao antígeno; (b) alteração de um ou mais nucleotídeos em um ou em ambos dos DNAs da etapa (a) que codificam os primeiros e segundos polipeptídeos para que haja uma maior diferença da capacidade de ligação à proteína A entre o primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e o segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou sem atividade de ligação ao antígeno; (c) introdução dos DNAs da etapa (b) em células hospedeiras e cultura das células hospedeiras para expressar os DNAs; e (d) coleta dos produtos de expressão da etapa (c) da cultura de células hospedeiras. [00096] Thus, the methods of the present invention for producing a polypeptide multimer can also be expressed as methods comprising the steps of: (a) supply of DNA encoding a first polypeptide having antigen binding activity and DNA encoding a second polypeptide having antigen binding activity or without antigen binding activity; (b) alteration of one or more nucleotides in one or both of the DNAs of step (a) that encode the first and second polypeptides so that there is a greater difference in the ability to bind to protein A between the first polypeptide having a binding activity the antigen and the second polypeptide having antigen-binding activity or without antigen-binding activity; (c) introducing the DNAs from step (b) into host cells and culturing the host cells to express the DNAs; and (d) collecting the expression products from step (c) of the host cell culture.
[0097] (a) fornecimento de DNA que codifica um primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e DNA que codifica um segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou sem atividade de ligação ao antígeno; (b) alteração de um ou mais nucleotídeos em um ou em ambos dos DNAs da etapa (a) que codificam os primeiros e segundos polipeptídeos para que haja uma maior diferença da capacidade de ligação à proteína A entre o primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e o segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou sem atividade de ligação ao antígeno; (c) introdução dos DNAs da etapa (b) em células hospedeiras e cultura das células hospedeiras para expressar os DNAs; e (d) coleta dos produtos de expressão da etapa (c) da cultura de células hospedeiras por cromatografia de afinidade em proteína A. [00097] The methods of the present invention for purifying a polypeptide multimer can also be expressed as methods comprising the step of: (a) supply of DNA encoding a first polypeptide having antigen binding activity and DNA encoding a second polypeptide having antigen binding activity or without antigen binding activity; (b) alteration of one or more nucleotides in one or both of the DNAs of step (a) that encode the first and second polypeptides so that there is a greater difference in the ability to bind to protein A between the first polypeptide having a binding activity the antigen and the second polypeptide having antigen-binding activity or without antigen-binding activity; (c) introducing the DNAs from step (b) into host cells and culturing the host cells to express the DNAs; and (d) collection of expression products from step (c) of host cell culture by protein A affinity chromatography.
[0098] [00098] In the present invention, a polypeptide multimer refers to a heteromeric multimer containing the first and the second polypeptide. It is preferred that the first and second polypeptides each have a different antigen-binding activity. The first and second polypeptides each having a different antigen-binding activity are not particularly limited while one of the polypeptides has a different antigen-binding domain (amino acid sequence) than that of another polypeptide. For example, as shown in Fig. 4 described below, a polypeptide can be fused to an antigen-binding domain that is different from that of another polypeptide. Alternatively, as shown in Figs 4, 6, and 9 described below, a polypeptide can be a polypeptide that monovalently binds to an antigen and lacks the antigen-binding domain possessed by another polypeptide. Polypeptide multimers containing such first and second polypeptides are also included in the polypeptide multimers of the present invention.
[0099] [00099] Multimers include dimers, trimers and tetramers, but are not limited to that.
[0100] [000100] In the present invention, a first polypeptide and / or a second polypeptide can form a multimer with one or two third polypeptides.
[0101] (a) expressão de um DNA que codifica um primeiro polipeptídeo tendo uma atividade de ligação ao antígeno, um DNA que codifica um segundo polipeptídeo tendo uma atividade de ligação ao antígeno, e um DNA que codifica dois terceiros polipeptídeos tendo uma atividade de ligação ao antígeno; e (b) coleta dos produtos de expressão da etapa (a); ou (a) expressão de um DNA que codifica um primeiro polipeptídeo tendo uma atividade de ligação ao antígeno, DNA que codifica um segundo polipeptídeo não tendo nenhuma atividade de ligação ao antígeno, e DNA que codifica um terceiro polipeptídeo tendo uma atividade de ligação ao antígeno; e (b) coleta dos produtos de expressão da etapa (a); em que um ou mais resíduos de aminoácidos em um ou tanto no primeiro polipeptídeo tendo uma atividade de ligação ao antígeno quanto no segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou sem atividade de ligação ao antígeno foram modificados para que haja uma maior diferença da capacidade de ligação à proteína A entre o primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e o segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou sem atividade de ligação ao antígeno.[000101] Accordingly, the present invention provides methods for producing a polypeptide multimer comprising a first polypeptide having antigen binding activity, a second polypeptide having antigen binding activity or without antigen binding activity, and one or two third polypeptides having antigen-binding activity, comprising the steps of: (a) expression of a DNA encoding a first polypeptide having an antigen binding activity, a DNA encoding a second polypeptide having an antigen binding activity, and a DNA encoding two third polypeptides having an antigen binding activity ; and (b) collecting the expression products from step (a); or (a) expression of a DNA encoding a first polypeptide having antigen binding activity, DNA encoding a second polypeptide having no antigen binding activity, and DNA encoding a third polypeptide having antigen binding activity; and (b) collecting the expression products from step (a); wherein one or more amino acid residues in one or both of the first polypeptide having antigen-binding activity and the second polypeptide having antigen-binding activity or without antigen-binding activity have been modified so that there is a greater difference in capacity of protein A binding between the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or without antigen binding activity.
[0102] (a) fornecimento de um DNA que codifica um primeiro polipeptídeo tendo uma atividade de ligação ao antígeno, um DNA que codifica um segundo polipeptídeo tendo uma atividade de ligação ao antígeno, e um DNA que codifica dois terceiros polipeptídeos tendo uma atividade de ligação ao antígeno; (b) alteração de um ou mais nucleotídeos em um ou em ambos dos DNAs da etapa (a) que codificam os primeiros e segundos polipeptídeos para que haja uma maior diferença da capacidade de ligação à proteína A entre o primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e o segundo polipeptídeo tendo uma atividade de ligação ao antígeno; (c) introdução dos DNAs que codificam o primeiro,segundo, e dois terceiros polipeptídeos em células hospedeiras, e cultura das células hospedeiras para expressar os DNAs; e (d) coleta dos produtos de expressão da etapa (c) da cultura de células hospedeiras; ou (a) fornecimento de um DNA que codifica um primeiro polipeptídeo tendo uma atividade de ligação ao antígeno, DNA que codifica um segundo polipeptídeo não tendo nenhuma atividade de ligação ao antígeno, e DNA que codifica um terceiro polipeptídeo tendo uma atividade de ligação ao antígeno; (b) alteração de um ou mais nucleotídeos em um ou em ambos dos DNAs da etapa (a) que codificam os primeiros e segundos polipeptídeos para que haja uma maior diferença da atividade de ligação à proteína A entre o primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e o segundo polipeptídeo não tendo nenhuma atividade de ligação ao antígeno; (c) introdução dos DNAs que codificam os primeiros, segundos, e terceiros polipeptídeos em células hospedeiras e cultura das células hospedeiras para expressar os DNAs; e (d) coleta dos produtos de expressão da etapa (c) da cultura de células hospedeiras. [000102] The methods described above can also be expressed as methods comprising the steps of: (a) providing a DNA encoding a first polypeptide having an antigen binding activity, a DNA encoding a second polypeptide having an antigen binding activity, and a DNA encoding two third polypeptides having an antigen binding activity ; (b) alteration of one or more nucleotides in one or both of the DNAs of step (a) that encode the first and second polypeptides so that there is a greater difference in the ability to bind to protein A between the first polypeptide having a binding activity the antigen and the second polypeptide having antigen-binding activity; (c) introducing the DNAs encoding the first, second, and two third polypeptides in host cells, and culturing the host cells to express the DNAs; and (d) collecting the expression products from step (c) of the host cell culture; or (a) providing a DNA encoding a first polypeptide having antigen binding activity, DNA encoding a second polypeptide having no antigen binding activity, and DNA encoding a third polypeptide having antigen binding activity; (b) alteration of one or more nucleotides in one or both of the DNAs of step (a) that encode the first and second polypeptides so that there is a greater difference in protein A binding activity between the first polypeptide having a binding activity the antigen and the second polypeptide having no antigen-binding activity; (c) introducing the DNAs that encode the first, second, and third polypeptides in host cells and culture of the host cells to express the DNAs; and (d) collecting the expression products from step (c) of the host cell culture.
[0103] [000103] Furthermore, in the present invention, the first and second polypeptides can form a multimer with third and fourth polypeptides.
[0104] (a) expressão de um DNA que codifica um primeiro polipeptídeo tendo uma atividade de ligação ao antígeno, um DNA que codifica um segundo polipeptídeo tendo uma atividade de ligação ao antígeno, e um DNA que codifica um terceiro polipeptídeo tendo uma atividade de ligação ao antígeno e um quarto polipeptídeo tendo uma atividade de ligação ao antígeno; e (b) coleta dos produtos de expressão da etapa (a); em que um ou mais resíduos de aminoácidos em um ou tanto no primeiro polipeptídeo tendo uma atividade de ligação ao antígeno quanto no segundo polipeptídeo tendo uma atividade de ligação ao antígeno foram modificados para que haja uma maior diferença da capacidade de ligação à proteína A entre o primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e o segundo polipeptídeo tendo uma atividade de ligação ao antígeno.[000104] Accordingly, the present invention provides methods for producing a polypeptide multimer comprising a first polypeptide having an antigen binding activity, a second polypeptide having an antigen binding activity, a third polypeptide having an antigen binding activity , and a fourth polypeptide having antigen-binding activity, comprising the steps of: (a) expression of a DNA encoding a first polypeptide having an antigen binding activity, a DNA encoding a second polypeptide having an antigen binding activity, and a DNA encoding a third polypeptide having an antigen binding activity and a fourth polypeptide having antigen-binding activity; and (b) collecting the expression products from step (a); wherein one or more amino acid residues in one or both of the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity have been modified so that there is a greater difference in protein A binding capacity between the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity.
[0105] (a) fornecimento de um DNA que codifica um primeiro polipeptídeo tendo uma atividade de ligação ao antígeno, um DNA que codifica um segundo polipeptídeo tendo uma atividade de ligação ao antígeno, e um DNA que codifica um terceiro polipeptídeo tendo uma atividade de ligação ao antígeno e um quarto polipeptídeo tendo uma atividade de ligação ao antígeno; (b) alteração de um ou mais nucleotídeos em um ou em ambos dos DNAs da etapa (a) que codificam os primeiros e segundos polipeptídeos para que haja uma maior diferença da capacidade de ligação à proteína A entre o primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e o segundo polipeptídeo tendo uma atividade de ligação ao antígeno; (c) introdução dos DNAs que codificam os primeiros, segundos, terceiros, e quartos polipeptídeos em células hospedeiras e cultura das células hospedeiras para expressar os DNAs; e (d) coleta dos produtos de expressão da etapa (c) da cultura de células hospedeiras. [000105] The methods described above can also be expressed as methods comprising the steps of: (a) providing a DNA encoding a first polypeptide having an antigen binding activity, a DNA encoding a second polypeptide having an antigen binding activity, and a DNA encoding a third polypeptide having an antigen binding activity and a fourth polypeptide having antigen-binding activity; (b) alteration of one or more nucleotides in one or both of the DNAs of step (a) that encode the first and second polypeptides so that there is a greater difference in the ability to bind to protein A between the first polypeptide having a binding activity the antigen and the second polypeptide having antigen-binding activity; (c) introducing the DNAs that encode the first, second, third, and fourth polypeptides in host cells and culture of the host cells to express the DNAs; and (d) collecting the expression products from step (c) of the host cell culture.
[0106] (a) expressão de um DNA que codifica um primeiro polipeptídeo tendo uma atividade de ligação ao antígeno, um DNA que codifica um segundo polipeptídeo tendo uma atividade de ligação ao antígeno, e um DNA que codifica dois terceiros polipeptídeos tendo uma atividade de ligação ao antígeno; e (b) coleta dos produtos de expressão da etapa (a); ou (a) expressão de um DNA que codifica um primeiro polipeptídeo tendo uma atividade de ligação ao antígeno, DNA que codifica um segundo polipeptídeo não tendo nenhuma atividade de ligação ao antígeno, e DNA que codifica um terceiro polipeptídeo tendo uma atividade de ligação ao antígeno; e (b) coleta dos produtos de expressão da etapa (a); em que um ou mais resíduos de aminoácidos em um ou tanto no primeiro polipeptídeo tendo uma atividade de ligação ao antígeno quanto no segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou sem atividade de ligação ao antígeno foram modificados para que haja uma maior diferença da capacidade de ligação à proteína A entre o primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e o segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou sem atividade de ligação ao antígeno.[000106] The present invention provides methods for purifying a polypeptide multimer comprising a first polypeptide having antigen binding activity, a second polypeptide having antigen binding activity or without antigen binding activity, and one or two third polypeptides having an antigen-binding activity, comprising the steps of: (a) expression of a DNA encoding a first polypeptide having an antigen binding activity, a DNA encoding a second polypeptide having an antigen binding activity, and a DNA encoding two third polypeptides having an antigen binding activity ; and (b) collecting the expression products from step (a); or (a) expression of a DNA encoding a first polypeptide having antigen binding activity, DNA encoding a second polypeptide having no antigen binding activity, and DNA encoding a third polypeptide having antigen binding activity; and (b) collecting the expression products from step (a); wherein one or more amino acid residues in one or both of the first polypeptide having antigen-binding activity and the second polypeptide having antigen-binding activity or without antigen-binding activity have been modified so that there is a greater difference in capacity of protein A binding between the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or without antigen binding activity.
[0107] (a) fornecimento de um DNA que codifica um primeiro polipeptídeo tendo uma atividade de ligação ao antígeno, um DNA que codifica um segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou sem atividade de ligação ao antígeno, e um DNA que codifica dois terceiros polipeptídeos tendo uma atividade de ligação ao antígeno; (b) alteração de um ou mais nucleotídeos em um ou em ambos dos DNAs da etapa (a) que codificam os primeiros e segundos polipeptídeos para que haja uma maior diferença da capacidade de ligação à proteína A entre o primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e o segundo polipeptídeo tendo uma atividade de ligação ao antígeno; (c) introdução dos DNAs que codificam o primeiro, segundo, e dois terceiros polipeptídeos em células hospedeiras e cultura das células hospedeiras para expressar os DNAs; e (d) coleta dos produtos de expressão da etapa (c) da cultura de células hospedeiras; ou (a) fornecimento de um DNA que codifica um primeiro polipeptídeo tendo uma atividade de ligação ao antígeno, DNA que codifica um segundo polipeptídeo não tendo nenhuma atividade de ligação ao antígeno, e DNA que codifica um terceiro polipeptídeo tendo uma atividade de ligação ao antígeno; (b) alteração de um ou mais nucleotídeos em um ou em ambos dos DNAs da etapa (a) que codificam os primeiros e segundos polipeptídeos para que haja uma maior diferença da capacidade de ligação à proteína A entre o primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e o segundo polipeptídeo não tendo nenhuma atividade de ligação ao antígeno; (c) introdução dos DNAs que codificam os primeiros, segundos, e terceiros polipeptídeos em células hospedeiras e cultura das células hospedeiras para expressar os DNAs; e (d) coleta dos produtos de expressão da etapa (c) da cultura de células hospedeiras. [000107] The methods described above can also be expressed as methods comprising the steps of: (a) providing a DNA encoding a first polypeptide having antigen binding activity, a DNA encoding a second polypeptide having antigen binding activity or without antigen binding activity, and a DNA encoding two third polypeptides having antigen-binding activity; (b) alteration of one or more nucleotides in one or both of the DNAs of step (a) that encode the first and second polypeptides so that there is a greater difference in the ability to bind to protein A between the first polypeptide having a binding activity the antigen and the second polypeptide having antigen-binding activity; (c) introducing the DNAs encoding the first, second, and two third polypeptides in host cells and culture of the host cells to express the DNAs; and (d) collecting the expression products from step (c) of the host cell culture; or (a) providing a DNA encoding a first polypeptide having antigen binding activity, DNA encoding a second polypeptide having no antigen binding activity, and DNA encoding a third polypeptide having antigen binding activity; (b) alteration of one or more nucleotides in one or both of the DNAs of step (a) that encode the first and second polypeptides so that there is a greater difference in the ability to bind to protein A between the first polypeptide having a binding activity the antigen and the second polypeptide having no antigen-binding activity; (c) introducing the DNAs that encode the first, second, and third polypeptides in host cells and culture of the host cells to express the DNAs; and (d) collecting the expression products from step (c) of the host cell culture.
[0108] (a) expressão de um DNA que codifica um primeiro polipeptídeo tendo uma atividade de ligação ao antígeno, um DNA que codifica um segundo polipeptídeo tendo uma atividade de ligação ao antígeno, um DNA que codifica um terceiro polipeptídeo tendo uma atividade de ligação ao antígeno, e um DNA que codifica um quarto polipeptídeo tendo uma atividade de ligação ao antígeno; e (b) coleta dos produtos de expressão da etapa (a) por cromatografia de afinidade em proteína A; em que um ou mais resíduos de aminoácidos em um ou tanto no primeiro polipeptídeo tendo uma atividade de ligação ao antígeno quanto no segundo polipeptídeo tendo uma atividade de ligação ao antígeno foram modificados para que haja uma maior diferença da capacidade de ligação à proteína A entre o primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e o segundo polipeptídeo tendo uma atividade de ligação ao antígeno.[000108] The present invention also provides methods for purifying a polypeptide multimer comprising a first polypeptide having an antigen binding activity, a second polypeptide having an antigen binding activity, a third polypeptide having an antigen binding activity, and a fourth polypeptide having antigen-binding activity, comprising the steps of: (a) expression of a DNA encoding a first polypeptide having an antigen binding activity, a DNA encoding a second polypeptide having an antigen binding activity, a DNA encoding a third polypeptide having an antigen binding activity, and a DNA encoding a fourth polypeptide having antigen-binding activity; and (b) collecting the expression products from step (a) by protein A affinity chromatography; wherein one or more amino acid residues in one or both of the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity have been modified so that there is a greater difference in protein A binding capacity between the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity.
[0109] (a) fornecimento de um DNA que codifica um primeiro polipeptídeo tendo uma atividade de ligação ao antígeno, um DNA que codifica um segundo polipeptídeo tendo uma atividade de ligação ao antígeno, um DNA que codifica um terceiro polipeptídeo tendo uma atividade de ligação ao antígeno, e um DNA que codifica um quarto polipeptídeo tendo uma atividade de ligação ao antígeno; (b) alteração de um ou mais nucleotídeos em um ou em ambos dos DNAs da etapa (a) que codificam os primeiros e segundos polipeptídeos para que haja uma maior diferença da capacidade de ligação à proteína A entre o primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e o segundo polipeptídeo tendo uma atividade de ligação ao antígeno; (c) introdução dos DNAs que codificam os primeiros, segundos, terceiros, e quartos polipeptídeos em células hospedeiras e cultura das células hospedeiras para expressar os DNAs; e (d) coleta dos produtos de expressão da etapa (c) da cultura de células hospedeiras por cromatografia de afinidade em proteína A. [000109] The methods described above can also be expressed as methods that comprise the steps of: (a) providing a DNA encoding a first polypeptide having an antigen binding activity, a DNA encoding a second polypeptide having an antigen binding activity, a DNA encoding a third polypeptide having an antigen binding activity, and a DNA encoding a fourth polypeptide having antigen-binding activity; (b) alteration of one or more nucleotides in one or both of the DNAs of step (a) that encode the first and second polypeptides so that there is a greater difference in the ability to bind to protein A between the first polypeptide having a binding activity the antigen and the second polypeptide having antigen-binding activity; (c) introducing the DNAs that encode the first, second, third, and fourth polypeptides in host cells and culture of the host cells to express the DNAs; and (d) collection of expression products from step (c) of host cell culture by protein A affinity chromatography.
[0110] [000110] In a polypeptide multimer of the present invention containing a first polypeptide, a second polypeptide, and one or two third polypeptides, the first and second polypeptides can each form a multimer (dimer) with the third polypeptide. In addition, two resulting dimers can form a multimer together. Two third polypeptides can have completely the same amino acid sequence (they can have binding activity to the same antigen). Alternatively, the third polypeptides can have the same amino acid sequence and two or more activities (for example, they can have binding activities to two or more different antigens). When only a third polypeptide is present, the third polypeptide can form a polypeptide multimer through dimerization with the first polypeptide or the second polypeptide.
[0111] [000111] In a polypeptide multimer of the present invention, the first and second polypeptides preferably have binding activity to different antigens. However, the third polypeptide may have binding activity to the same antigen as that of one or both of the first and second polypeptides. Alternatively, the third polypeptide may have antigen-binding activity different from that of the first and second polypeptides.
[0112] [000112] Alternatively, a polypeptide multimer of the present invention can contain a first polypeptide, second polypeptide, third polypeptide, and fourth polypeptide. In such a polypeptide multimer, the first polypeptide and the second polypeptide can form a multimer (dimer) with the third polypeptide and fourth polypeptide, respectively. For example, by forming disulfide bonds in the medium, the first polypeptide and the third polypeptide can form a dimer, and the second polypeptide and the fourth polypeptide can form a dimer.
[0113] [000113] In a polypeptide multimer of the present invention, the first and second polypeptides preferably have binding activity to different antigens. However, the third polypeptide may have binding activity to the same antigen as that of one or both of the first and second polypeptides. Alternatively, the third polypeptide may have antigen-binding activity different from that of the first and second polypeptides. In addition, the fourth polypeptide may have binding activity to the same antigen as that of one or both of the first and second polypeptides. Alternatively, the fourth polypeptide may have antigen-binding activity different from that of the first and second polypeptides.
[0114] [000114] Specifically, for example, when the first and second polypeptides contain the amino acid sequence of an antibody heavy chain against antigen A and the amino acid sequence of an antibody heavy chain against antigen B, respectively, the third and fourth polypeptides may contain the amino acid sequence of an antibody light chain against antigen A and the amino acid sequence of an antibody light chain against antigen B, respectively. When a polypeptide multimer of the present invention has third and fourth polypeptides that contain two different antibody light chain amino acid sequences, a highly pure polypeptide multimer of interest can be efficiently produced or purified by making the pi values of the third and fourth polypeptide different using the methods described below, or differentiating their ability to bind protein L, in addition to differentiating the ability to bind protein A between the first and second polypeptides.
[0115] [000115] Alternatively, for example, when the first polypeptide has the amino acid sequence of an antibody heavy chain against antigen A, the second polypeptide has the amino acid sequence of a variable region of the antibody light chain against antigen B and the amino acid sequence of an antibody heavy chain constant region, the third polypeptide has the amino acid sequence of an antibody light chain against antigen A, and the fourth polypeptide has the amino acid sequence of a variable region of the heavy chain of antibody against B antigen and the amino acid sequence of an antibody light chain constant region, a highly pure polypeptide multimer of interest having the first, second, third, and fourth polypeptides can also be efficiently produced or purified using the present invention. In this case, as described in Example 12 below, introducing amino acid mutations to change the pi value of a polypeptide or introducing amino acid mutations to promote the association of polypeptides of interest (WO2006 / 106905) allows for more efficient purification or producing a polypeptide multimer of interest having the first, second, third and fourth polypeptide in higher purity. Amino acid mutations to be introduced to promote the association of polypeptides can be those used in the methods described in Protein Eng. 1996 Jul., 9 (7): 617-21; Protein Eng Des Sel. 2010 Apr., 23 (4): 195-202; J Biol Chem. 2010 Jun. 18, 285 (25): 19637-46; WO2009080254; and such, in which two polypeptides having a heavy chain constant region are associated heteromerically by modifying the CH3 domain of the heavy chain constant region; and those used in the methods described in WO2009080251, WO2009080252, WO2009080253, and such, by which the association of a particular heavy and light chain pair is promoted.
[0116] [000116] In the present invention, "polypeptide having antigen-binding activity" refers to a peptide or protein of five or more amino acids in length having a domain (region) capable of binding to a protein or peptide, such as a antigen or linker, for example, a variable region of the heavy chain or light chain of the antibody, receptor, Fc-receptor domain fusion peptide, structure, or a fragment thereof. Specifically, a polypeptide having antigen-binding activity can contain the amino acid sequence of an antibody variable region, receptor, Fc-receptor domain fusion peptide, structure, or a fragment thereof.
[0117] [000117] The structure can be any polypeptide as long as it is a conformationally stable polypeptide capable of binding to at least one antigen. Such polypeptides include, but are not limited to, for example, fragments of antibody variable region, fibronectin, protein A domains, LDL receptor A domains, lipocalins, and molecules mentioned in Nygren et al. (Current Opinion in Structural Biology, 7: 463-469 (1997); Journal of Immunol. Methods, 290: 3-28 (2004)), Binz et al. (Nature Biotech 23: 1257-1266 (2005)), and Hosse et al. (Protein Science 15: 14-27 (2006))
[0118] [000118] Methods for obtaining variable regions of antibody, receptors, Fc-receptor domain fusion peptides, structure, and fragments thereof are known to those skilled in the art.
[0119] [000119] Such polypeptides having antigen-binding activity can be derived from a living organism or artificially designed. Polypeptides can be derived from natural proteins, synthetic proteins, recombinant proteins, and the like. In addition, polypeptides can be peptides or protein fragments of 10 or more amino acids in length that have a domain (region) capable of binding to a protein or peptide, such as an antigen or linker, while having the ability to bind to an antigen. Polypeptides can have more than one domain capable of binding to an antigen (including the ligand).
[0120] [000120] A polypeptide having antigen-binding activity can also be referred to as a polypeptide having a protein antigen-binding domain (s).
[0121] [000121] In the present invention, "polypeptide having no antigen binding activity" refers to a peptide or protein of five or more amino acids in length, such as an antibody fragment having no antigen binding activity, domain of Fc, structure, or a fragment thereof. Specifically, a polypeptide having no antigen-binding activity can contain the amino acid sequence of an antibody constant region, Fc domain, structure, or fragment thereof, but the amino acid sequence is not limited to the examples above. A polypeptide having no antigen binding activity can be combined with a polypeptide having antigen binding activity to produce a polypeptide multimer that monovalently binds to an antigen.
[0122] [000122] In the present invention, the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or without antigen binding activity can contain the amino acid sequence of a constant region of the antibody heavy chain or amino acid sequence of an antibody Fc domain. The amino acid sequence of an antibody Fc domain or an antibody constant region heavy chain includes, but is not limited to, those of human IgG-like constant regions and Fc domains. Constant regions like IgG or Fc domains can be natural isotype IgG1, IgG2, IgG3, or IgG4, or can be variants of them.
[0123] [000123] However, in the present invention, the third polypeptide having an antigen binding activity and the fourth polypeptide having an antigen binding activity can contain the amino acid sequence of a constant region of the antibody light chain. The amino acid sequence of an antibody light chain constant region includes, but is not limited to, those of human kappa and human lambda constant regions, and variants thereof.
[0124] [000124] In addition, in the present invention, polypeptides having antigen-binding activity may contain the amino acid sequence of an antibody variable region (for example, the amino acid sequences of CDR1, CDR2, CDR3, FR1, FR2, FR3 and FR4).
[0125] [000125] Furthermore, in the present invention, polypeptides having antigen-binding activity can contain the amino acid sequence of an antibody heavy chain or an antibody light chain. More specifically, the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or without antigen binding activity can contain the amino acid sequence of an antibody heavy chain. However, the third polypeptide having antigen-binding activity and the fourth polypeptide having antigen-binding activity can contain the amino acid sequence of an antibody light chain.
[0126] [000126] When a polypeptide multimer of interest is a tetramer that is formed by multimerization between a dimer formed by the first and third polypeptide and a dimer formed by the second and fourth polypeptide, for example, a polypeptide in which the first and second polypeptide having a antigen binding activity contains the amino acid sequence of an antibody heavy chain, and a polypeptide in which the third and fourth polypeptide having antigen binding activity contain the amino acid sequence of an antibody light chain, can be used to the polypeptide multimer of the present invention. Alternatively, a polypeptide in which the first polypeptide having antigen binding activity contains the amino acid sequence of an antibody heavy chain, a polypeptide in which the second polypeptide having antigen binding activity contains the amino acid sequence of a region antibody light chain variable and the amino acid sequence of an antibody heavy chain constant region, a polypeptide in which the third polypeptide having antigen binding activity contains the amino acid sequence of an antibody light chain, and a polypeptide in which the fourth polypeptide having antigen-binding activity contains the amino acid sequence of a variable region of the antibody heavy chain, can also be used.
[0127] [000127] Specifically, a polypeptide multimer of the present invention can be a multispecific antibody.
[0128] [000128] In the present invention, "a multispecific antibody" refers to an antibody capable of specifically binding to at least two different antigens.
[0129] [000129] In the present invention, "different antigens" refer not only to different antigen molecules per se, but also to different antigen determinants present in the same antigen molecules. Consequently, for example, different antigen determinants present within a single molecule are included in the "different antigens" of the present invention. In the present invention, antibodies that recognize several different antigen determinants in a single molecule are considered to be "antibodies capable of specifically binding to different antigens".
[0130] [000130] In the present invention, multispecific antibodies include, but are not limited to, bispecific antibodies capable of specifically binding two types of antigens. Preferred bispecific antibodies of the present invention include IgG type H2L2 antibodies (composed of two types of H chains and two types of L chains) having a human IgG constant region. More specifically, such antibodies include, but are not limited to, for example, chimeric IgG antibodies, humanized antibodies and human antibodies.
[0131] [000131] In addition, a polypeptide having antigen-binding activity can be, for example, a molecule in which at least two of a variable region of the heavy chain, variable region of the light chain, constant region of the heavy chain, and region constant in the light chain, are linked together as a single chain. Alternatively, the polypeptide can be an antibody in which at least two of a heavy chain variable region, light chain variable region, Fc domain (constant region without CH1 domain), and light chain constant region, are linked together as a single chain.
[0132] [000132] In the present invention, the phrase "there is a greater difference in protein A binding capacity between polypeptides having antigen binding activity" means that protein A binding capacity is not the same (it is different) between two or more polypeptides as a result of modifications of amino acids on the surface of polypeptides having antigen-binding activity. More specifically, this phrase means that, for example, the protein A's binding capacity of the first polypeptide having an antigen binding activity is different from that of the second polypeptide having an antigen binding activity. The difference in protein A binding capacity can be examined, for example, using protein A affinity chromatography.
[0133] [000133] The strength of the protein A binding capacity of a polypeptide having an antigen binding activity is correlated with the pH of the solvent used for the elution. The greater the protein A binding capacity of the polypeptide is, the lower the pH of the solvent used for elution becomes. Thus, the phrase "there is a greater difference in protein A binding capacity between polypeptides having antigen binding activity" can also be expressed as "when two or more polypeptides having antigen binding activity are eluted using chromatography of protein A affinity, each polypeptide is eluted at a different solvent pH ". The difference in pH of the eluting solvent is 0.1 or more, preferably 0.5 or more, and even more preferably 1.0 or more, but is not limited to this.
[0134] [000134] Furthermore, in the present invention, it is preferable to alter the protein A binding capacity without reducing other activities (e.g., plasma retention) of the polypeptides having an antigen binding activity.
[0135] - um anticorpo homomérico compreendendo duas unidades da primeira cadeia pesada do anticorpo e duas unidades da cadeia L comum - um anticorpo biespecífico compreendendo a primeira cadeia pesada do anticorpo, a segunda cadeia pesada do anticorpo, e duas unidades da cadeia L comum - um anticorpo homomérico compreendendo duas unidades da segunda cadeia pesada do anticorpo e duas unidades da cadeia L comum [000135] A polypeptide multimer of interest comprising the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or without antigen binding activity can be produced or purified using protein A affinity chromatography based on the difference in protein A binding capacity between the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or without antigen binding activity. Specifically, for example, when the polypeptide multimer of the present invention is a bispecific antibody that has a common L chain (i.e., the same amino acid sequence in the third and fourth polypeptides), the polypeptide multimer can be produced or purified by the method described below . First, host cells are introduced with the following: a nucleic acid encoding the first polypeptide having antigen-binding activity (more specifically, the antibody's first heavy chain) whose amino acid at position 435 (EU number) in the sequence of amino acids in the antibody heavy chain constant region is arginine (R); a nucleic acid encoding the second polypeptide having antigen-binding activity (more specifically, the second heavy chain of the antibody) whose amino acid at position 435 (EU numbering) in the amino acid sequence of the constant region of the heavy chain of the antibody is histidine ( H); and a nucleic acid encoding the third polypeptide having antigen-binding activity (common L chain). The cells are grown to express DNA transiently. Then, the resulting expression products are loaded onto a protein A column. After washing, elution is carried out first with a high pH elution solution and then with a low pH elution solution. A homomeric antibody comprising two units of the first antibody heavy chain and two units of the common L chain has no protein A binding site in its constant region of the heavy chain. However, a bispecific antibody comprising the antibody first heavy chain, the antibody second heavy chain, and two units of the common L chain has a unique protein A binding site in its heavy chain constant region. A homomeric antibody comprising two units of the second antibody heavy chain and two units of the common L chain has two protein A binding sites in its constant region of the heavy chain. As described above, the protein A's binding capacity of a polypeptide is correlated with the pH of solvent to elute the polypeptide in protein A affinity chromatography. The greater the protein A binding capacity is, the lower the solvent pH of the elution becomes. Thus, when the elution is carried out first with a high pH elution solution and then with a low pH elution solution, the antibodies are eluted in the following order: - a homomeric antibody comprising two units of the first antibody heavy chain and two units of the common L chain - a bispecific antibody comprising the first antibody heavy chain, the second antibody heavy chain, and two units of the common L chain - a homomeric antibody comprising two units of the second antibody heavy chain and two units of the common L chain
[0136] [000136] This allows the production or purification of the polypeptide multimers (bispecific antibodies) of interest.
[0137] [000137] The purity of the polypeptide multimers obtained by the purification or production methods of the present invention is at least 95% or greater (for example, 96%, 97%, 98%, 99% or greater).
[0138] (1) modificação de um ou mais resíduos de aminoácidos na sequência de aminoácidos de um do primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e do segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou de nenhuma atividade de ligação ao antígeno, tal que a capacidade de ligação à proteína A de um dos polipeptídeos é aumentada; (2) modificação de um ou mais resíduos de aminoácidos na sequência de aminoácidos de um do primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e do segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou de nenhuma atividade de ligação ao antígeno, tal que a capacidade de ligação à proteína A de um dos polipeptídeos é reduzida; e (3) modificação de um ou mais resíduos de aminoácidos no primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e no segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou sem atividade de ligação ao antígeno, tal que a capacidade de ligação à proteína A de um do primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e do segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou de nenhuma atividade de ligação ao antígeno é aumentada, e a capacidade de ligação à proteína A de outro polipeptídeo é reduzida. [000138] Amino acid residue modifications to create a difference in protein A binding capacity between the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or without antigen binding activity include , but are not limited to: (1) modification of one or more amino acid residues in the amino acid sequence of one of the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or no antigen binding activity, such that the protein A's binding capacity of one of the polypeptides is increased; (2) modification of one or more amino acid residues in the amino acid sequence of one of the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or no antigen binding activity, such that the protein A's binding capacity of one of the polypeptides is reduced; and (3) modification of one or more amino acid residues in the first polypeptide having antigen binding activity and in the second polypeptide having antigen binding activity or without antigen binding activity, such that the protein A binding capacity of one of the first polypeptide having antigen-binding activity and the second polypeptide having antigen-binding activity or no antigen-binding activity is increased, and the protein A-binding capacity of another polypeptide is reduced.
[0139] [000139] In the present invention, it is preferred that amino acids on the surface of a polypeptide having antigen binding activity or without antigen binding activity are modified. In addition, it is also preferred to consider reducing the influence of the modification on other activities of the polypeptide.
[0140] [000140] Consequently, in the present invention, it is preferable to modify, for example, amino acid residues at the following positions (EU numbering) in the Fc domain or constant region of the antibody heavy chain: TLMISR at positions 250-255, VLHQDWLNGK at positions 308-317, and EALHNHY at positions 430-436; preferably, TLMIS in positions 250-254, LHQD in positions 309-312, LN in positions 314 and 315, E in position 430, and LHNHY in positions 432-436; more preferably, LMIS at positions 251-254, LHQ at positions 309-311, L at position 314, and LHNH at positions 432435; and especially, informational steering system in positions 252-254, L in position 309, Q in position 311, and NHY in positions 434-436.
[0141] [000141] As for amino acid modifications of the variable region of the antibody heavy chain, the preferred mutation sites include FR1, CDR2 and FR3. The most preferred mutation sites include, for example, positions H15-H23, H56-H59, H63-H72, and H79-H83 (EU numbering).
[0142] [000142] Of the above amino acid modifications, modifications that do not reduce binding to FcRn or plasma retention in transgenic mice with human FcRn are more preferred.
[0143] [000143] More specifically, modifications that increase protein A binding capacity of a polypeptide include, but are not limited to, the replacement of histidine (His) of the amino acid residue at position 435 (EU numbering) in the amino acid sequence of a antibody Fc domain or an antibody heavy chain constant region.
[0144] [000144] However, modifications that reduce the protein A binding capacity of a polypeptide include, but are not limited to, substitution of the arginine of the amino acid residue at position 435 (EU numbering) in the amino acid sequence of an antibody or Fc domain a constant region of the antibody heavy chain.
[0145] [000145] As for the variable region of the antibody heavy chain, the variable region of the heavy chain of the VH3 subclass has protein A binding activity. Thus, to increase protein A binding capacity, amino acid sequences at the modification sites above are preferably identical to those of the variable region of the VH3 subclass heavy chain. To reduce the binding capacity of protein A, the amino acid sequences are preferably identical to those of the variable region of the heavy chain of another subclass.
[0146] [000146] As described below, modification of amino acid residues can be achieved by altering one or more nucleotides in a DNA encoding a polypeptide, and expressing the DNA in host cells. Those skilled in the art can readily determine the number, site, and type of nucleotides altered depending on the type of amino acid residues after modification.
[0147] [000147] In this application, modification (alteration) refers to the substitution, deletion, addition or insertion, or combinations thereof.
[0148] - modificação de aminoácido para aumentar a taxa da associação heteromérica de dois tipos de cadeias H em um anticorpo biespecífico - modificação de aminoácido para estabilizar as ligações dissulfeto entre o primeiro polipeptídeo tendo uma atividade de ligação ao antígeno e o segundo polipeptídeo tendo uma atividade de ligação ao antígeno ou sem atividade de ligação ao antígeno - modificação de aminoácido para melhorar a retenção plasmática de um anticorpo - modificação para aumentar a estabilidade sob condições acídicas - modificação para reduzir a heterogeneidade - modificação para suprimir a reação de deamidação - modificação para introduzir uma diferença entre os pontos isoelétricos de dois tipos de polipeptídeos - modificação para alterar a capacidade de ligação pelo receptor γ [000148] The polypeptide having antigen-binding activity may comprise other modifications in addition to the above amino acid residue modifications. Such additional modifications can be selected from, for example, amino acid substitutions, deletions and modifications, and combinations thereof. Specifically, all polypeptides whose amino acid sequences comprise a modification described below are included in the present invention: - amino acid modification to increase the rate of heteromeric association of two types of H chains in a bispecific antibody - amino acid modification to stabilize disulfide bonds between the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or without antigen binding activity - amino acid modification to improve the plasma retention of an antibody - modification to increase stability under acidic conditions - modification to reduce heterogeneity - modification to suppress the walking reaction - modification to introduce a difference between the isoelectric points of two types of polypeptides - modification to change the binding capacity by the γ receptor
[0149] [000149] These amino acid modifications are described below. Amino acid modification to increase the rate of heteromeric association between two types of H chains in a bispecific antibody
[0150] [000150] The amino acid modifications of the present invention can be combined with the amino acid modifications described in WO2006106905. There is no limitation at the sites of modification while amino acids form the interface between two polypeptides having antigen-binding activity. Specifically, for example, when a heavy chain constant region is modified, such modifications include modifications that make the amino acids of at least one of the combinations of positions 356 and 439, positions 357 and 370, and positions 399 and 409 (EU numbering) in the amino acid sequence of the heavy chain constant region of the first polypeptide having an antigen-binding activity having the same electrical charge; and amino acids from at least one of the combinations of positions 356 and 439, positions 357 and 370, and positions 399 and 409 (EU numbering) in the heavy chain constant region of the second polypeptide having antigen binding activity or without binding activity to the antigen have an electrical charge as opposed to that of the first polypeptide having an antigen-binding activity. More specifically, such modifications include, for example, the introduction of a mutation that replaces Glu at position 356 (EU numbering) with Lys in the amino acid sequence of the heavy chain constant region of any of the first polypeptide having antigen-binding activity and of the second polypeptide having antigen-binding activity, and a mutation that replaces Lys at position 439 (EU numbering) with Glu in the amino acid sequence of the heavy chain constant region of another polypeptide. When these modifications are combined with the modifications of the present invention, the polypeptide of interest can be obtained in a higher purity by purification based on protein A alone.
[0151] [000151] Alternatively, the polypeptide multimer of interest comprising the first, second, third and fourth polypeptide having an antigen binding activity can be efficiently produced or purified to a higher purity, when the modification is carried out to produce the amino acids in the position 39 (Kabat numbering) in the variable region of the heavy chain and / or position 213 (EU numbering) in the heavy chain constant region of the first polypeptide having an antigen binding activity has an electrical charge as opposed to that of the amino acid at position 39 (Kabat numbering) in the variable region of the heavy chain and / or the amino acid at position 213 (EU numbering) in the constant region of the heavy chain of the second polypeptide having antigen-binding activity or without antigen-binding activity, and the amino acid in position 38 (Kabat numbering) and / or the amino acid at position 123 (EU numbering) in the variable region of the light chain of the third polypeptide having a antigen-binding activity has an electrical charge as opposed to that of the amino acid at position 38 (Kabat numbering) and / or the amino acid at position 123 (EU numbering) in the variable region of the fourth polypeptide light chain having antigen binding activity. Amino acid modification to stabilize disulfide bonds between the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or without antigen binding activity
[0152] [000152] As described in published documents (Mol. Immunol. 1993, 30, 105-108; and Mol. Immunol. 2001, 38, 1-8), the heterogeneity of IgG4 is eliminated and its stable structure can be maintained by replacing Pro for Being at position 228 (EU numbering) in the amino acid sequence of the IgG4 heavy chain constant region. Amino acid modification to improve plasma retention of an antibody
[0153] [000153] In order to regulate plasma retention, it is possible to combine the amino acid modifications of the present invention with amino acid modifications that alter the pI value of the antibody. Modifications to constant regions include, for example, amino acid changes at positions 250 and 428 (EU numbering) and as described in published documents (J. Immunol. 2006, 176 (1): 346-356; and Nat. Biotechnol. 1997 15 (7): 637-640). Modifications to variable regions include amino acid modifications described in WO2007 / 114319 and WO2009 / 041643. The amino acids to be modified are preferably exposed on the surface of a polypeptide having antigen-binding activity. The modifications include, for example, the amino acid substitution at position 196 (EU numbering) in the amino acid sequence of a constant region of the heavy chain. In the case of the IgG4 heavy chain constant region, plasma retention can be increased, for example, by replacing glutamine with lysine at position 196 thereby reducing the pI value.
[0154] [000154] In addition, plasma retention can be regulated by changing the ability to bind to FcRn. Amino acid modifications that alter the ability to bind to FcRn include, for example, amino acid substitutions in the constant region of the antibody heavy chain described in published documents (The Journal of Biological Chemistry vol.276, No.9 6591-6604, 2001; Molecular Cell, Vol.7, 867-877, 2001; Curr Opin Biotechnol. 2009, 20 (6): 685-91). Such amino acid substitutions include, for example, substitutions at positions 233, 238, 253, 254, 255, 256, 258, 265, 272, 276, 280, 285, 288, 290, 292, 293, 295, 296, 297, 298, 301, 303, 305, 307, 309, 311, 312, 315, 317, 329, 331, 338, 360, 362, 376, 378, 380, 382, 415, 424, 433, 434, 435 and 436 ( EU numbering). Modification to improve stability under acidic conditions
[0155] [000155] When the IgG4 heavy chain constant region is used, the stable four-chain structure (H2L2 structure) is preferably maintained by suppressing the conversion of IgG4 as a half-molecule under acidic conditions. Thus, arginine at amino acid position 409 (EU numbering system) that plays an important role in maintaining the four-chain structure (Immunology 2002, 105, 9-19) is preferably replaced by lysine of the IgG1 type which maintains a structure of four chains stable even under acidic conditions. In addition, to improve the acidic stability of IgG2, methionine at amino acid position 397 (EU numbering system) can be replaced by valine. These modifications can be used in combination with the amino acid modifications of the present invention. Modification to reduce heterogeneity
[0156] [000156] The amino acid modifications of the present invention can be combined with the methods described in WO2009041613. Specifically, for example, the modification in which the two amino acids at the C-terminus of the IgG1 heavy chain constant region (ie, glycine and lysine at positions 446 and 447 [EU numbering], respectively) can be combined with the amino acid modifications described in the Examples in this application. Modification to suppress the ambulation reaction
[0157] [000157] The amino acid modifications of the present invention can be combined with amino acid modifications to suppress the walking reaction. The walking reaction has been reported to occur more frequently at a site where asparagine (N) and glycine (G) are adjacent to each other (--- NG ---) (Geiger et al., J. Bio. Chem. (1987 ) 262: 785-794). When a polypeptide multimer (multispecific antibody) of the present invention has a site where asparagine and glycine are adjacent to each other, the walking reaction can be suppressed by modifying the amino acid sequence. Specifically, for example, either asparagine or glycine is replaced by other amino acids. More specifically, for example, asparagine is replaced with aspartic acid. Modification to introduce a difference in the isoelectric point between two types of polypeptides
[0158] [000158] The amino acid modifications of the present invention can be combined with amino acid modifications to introduce a difference in the isoelectric point. Specific methods are described, for example, in WO2007 / 114325. In addition to the modifications of the present invention, the amino acid sequences of the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or without antigen binding activity are modified so that there is a greater difference in the isoelectric point among these polypeptides. This allows for efficient production or purification of the polypeptide of interest to a higher purity. In addition, a greater difference in the isoelectric point can be produced between the third polypeptide having an antigen binding activity and the fourth polypeptide having an antigen binding activity. This allows the polypeptide multimer of interest comprising the first, second, third and fourth polypeptide to be efficiently produced or purified to a higher purity. Specifically, when the first and second polypeptides each comprise an amino acid sequence of an antibody heavy chain, the sites of modification include, for example, positions 1, 3, 5, 8, 10, 12, 13, 15, 16 , 19, 23, 25, 26, 39, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 81, 82b, 83, 85, 86, 105, 108, 110 and 112 (numbering Kabat). When the third and fourth polypeptides each comprise an amino acid sequence of an antibody light chain, the sites of modification include, for example, positions 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 38, 39, 41,42, 43, 45, 46, 49, 57, 60, 63, 65, 66, 68, 69, 70, 74, 76, 77, 79, 80, 81, 85, 100, 103, 105, 106, 107 and 108 (Kabat numbering). A major difference in the isoelectric point can be produced by modifying at least one of the amino acid residues in the above positions in one polypeptide to have an electric charge, and by modifying at least one of the amino acid residues in the above positions in another polypeptide not to have any charge or electrical charge opposite to the above. Modification to change the ability to bind to the Fcγ receptor
[0159] [000159] The amino acid modifications of the present invention can be combined with amino acid modifications that alter (increase or reduce) the Fcγ receptor binding capacity. Modifications to alter the ability to bind to the Fcγ receptor include, but are not limited to, the modifications described in Curr Opin Biotechnol. 2009, 20 (6): 685-91. Specifically, the ability to bind to the Fcγ receptor can be altered, for example, by combining the modifications of the present invention with a modification that replaces leucine at positions 234 and 235 and asparagine at position 272 (EU numbering) of a constant region of the heavy chain IgG1 by other amino acids. Amino acids after substitution include, but are not limited to, alanine.
[0160] [000160] The preparation of DNAs that encode polypeptides having antigen binding activity, modification of one or more nucleotides, expression of a DNA, and recovery of expression products is described below. Preparation of DNAs that encode polypeptides having antigen-binding activity
[0161] [000161] In the present invention, a DNA encoding a polypeptide having antigen binding activity or a polypeptide having no antigen binding activity can be the total or a portion of a known sequence (naturally occurring or artificial sequence) , or combinations thereof. Such DNAs can be obtained by methods known to those skilled in the art. DNAs can be isolated, for example, from antibody libraries, or by cloning the genes encoding hybridoma antibodies that produce monoclonal antibodies.
[0162] [000162] As for antibody libraries, many are already well known, and those skilled in the art can appropriately obtain antibody libraries since methods for producing antibody libraries are known. For example, for antibody phage libraries, it can refer to the literature, such as Clackson et al., Nature 1991, 352: 624-8; Marks et al., J. Mol. Biol. 1991, 222: 581-97; Waterhouses et al., Nucleic Acids Res. 1993, 21: 2265-6; Griffiths et al., EMBO J. 1994, 13: 3245-60; Vaughan et al., Nature Biotechnology 1996, 14: 309-14; or Japanese Patent Publication Kohyo No. (JP-A) H20-504970 (unexamined Japanese national phase publication corresponding to a non-Japanese international publication). In addition, known methods, such as methods that use eukaryotic cells as libraries (WO95 / 15393) and ribosome exposure methods can be used. In addition, techniques for obtaining human antibodies by separation using human antibody libraries are also known. For example, variable regions of human antibodies can be expressed on the phage surface as single chain antibodies (scFvs) using phage exposure methods, and phages that bind to antigens can be selected. Genetic analysis of the selected phages can determine the DNA sequences that encode the variable regions of human antibodies that bind to the antigens. Once the DNA sequences of scFvs that bind to the antigens are revealed, suitable expression vectors can be produced based on these sequences to obtain human antibodies. These methods are already well known, and can refer to WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438 and WO95 / 15388.
[0163] [000163] As for methods for obtaining genes that encode hybridoma antibodies, basically, known techniques can be used. Specifically, desired antigens or cells expressing the desired antigens are used as sensitizing antigens for immunization according to conventional immunization methods. The immune cells obtained in this way are fused with parental cells known by ordinary cell fusion methods, and the monoclonal antibody that produces cells (hybridomas) is screened by ordinary screening methods. cDNAs from antibody variable regions (V regions) can be obtained by reverse transcribing mRNAs from hybridomas obtained using reverse transcriptase. Antibody encoding genes can be obtained by linking them with DNAs that encode the desired antibody constant regions (C regions).
[0164] [000164] More specifically, without limitations, the following methods are examples.
[0165] [000165] Sensitizing antigens for obtaining antibody genes encoding antibody heavy and light chains include both antigens complete with immunogenicity and incomplete antigens composed of haptens and the like that do not show antigenicity. For example, complete proteins and partial peptides of proteins of interest can be used. In addition, it is known that substances composed of polysaccharides, nucleic acids, lipids, and the like can become antigens. Accordingly, there are no particular limitations on antigens in the present invention. Antigens can be prepared by methods known to those skilled in the art, and can be prepared, for example, by the following methods using baculovirus (for example, WO98 / 46777). Hybridomas can be produced, for example, by the following methods by Milstein et al. (G. Kohler and C. Milstein, Methods Enzymol. 1981, 73: 3-46), and the like. When the immunogenicity of an antigen is low, it can be linked to a macromolecule that has the immunogenicity, such as albumin, and then used for immunization. In addition, by linking antigens with other molecules if necessary, they can be converted into soluble antigens. When transmembrane molecules, such as receptors, are used as antigens, portions of the extracellular regions of the receptors can be used as a fragment, or cells that express transmembrane molecules on their cell surface can be used as immunogens.
[0166] [000166] Antibody-producing cells can be obtained by immunizing animals using the appropriate sensitizing antigens described above. Alternatively, antibody-producing cells can be prepared by in vitro immunization of lymphocytes that can produce antibodies. Various mammals can be used as animals for immunization, where rodents, lagomorphs and primates are generally used. Examples of such animals include mice, rats, and rodent hamsters, rabbits for lagomorphs, and monkeys including cinomolgus monkeys, rhesus monkeys, baboons and primate chimpanzees. In addition, transgenic animals that carry gene repertoires of human antibodies are also known, and human antibodies can be obtained using these animals (see WO96 / 34096; Mendez et al., Nat. Genet. 1997, 15: 14656). Instead of using such transgenic animals, for example, desired human antibodies having binding activity against antigens can be obtained by sensitizing in vitro human lymphocytes to desired antigens or cells that express the desired antigens, and then fusing the sensitized lymphocytes with cells of human myeloma, such as U266 (see Japanese Patent Application Publication Kokoku No. (JP-B) H1-59878 (examined, published Japanese patent application approved for opposition)). In addition, the desired human antibodies can be obtained by immunizing transgenic animals that carry a complete repertoire of human antibody genes, with desired antigens (see WO93 / 12227, WO92 / 03918, WO94 / 02602, WO96 / 34096 and WO96 / 33735).
[0167] [000167] Animal immunization can be performed by appropriately diluting and suspending a sensitizing antigen in Phosphate-Buffered Saline (PBS), physiological saline, or similar, and forming an emulsion by mixing an adjuvant if necessary, followed by an intraperitoneal or subcutaneous injection into the animals. Then, the sensitizing antigen mixed with Freund's incomplete adjuvant is preferably administered several times every four to 21 days. Antibody production can be confirmed by measuring the target antibody titer in animal sera using conventional methods.
[0168] [000168] Antibody-producing cells obtained from lymphocytes or animals immunized with a desired antigen can be fused with myeloma cells to generate hybridomas using conventional fusion agents (eg, polyethylene glycol) (Goding, Antibodies: Principles and Practice Monoclonal, Academic Press , 1986, 59-103). When necessary, hybridoma cells can be cultured and developed, and the binding specificity of the antibody produced from these hybridomas can be measured using known methods of analysis, such as immunoprecipitation, radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA). Next, hybridomas that produce antibodies of interest whose specificity, affinity or activity have been determined can be subcloned by methods, such as dilution limitation.
[0169] [000169] Next, the genes encoding the selected antibodies can be cloned from hybridomas or antibody-producing cells (sensitized lymphocytes, and the like) using probes that can specifically bind to the antibodies (for example, oligonucleotides complementary to sequences that encode antibody constant regions). Cloning of the mRNA using RT-PCR is also possible. Immunoglobulins are classified into five different classes, IgA, IgD, IgE, IgG and IgM. These classes are further divided into several subclasses (isotypes) (for example, IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2; and the like). The heavy chains and light chains used in the present invention to produce antibodies are not particularly limited and can be derived from antibodies that belong to any of these classes or subclasses; however, IgG is particularly preferred.
[0170] [000170] In this application, it is possible to modify genes that encode the heavy chain and genes that encode the light chain using genetic engineering techniques. Genetically modified antibodies, such as chimeric antibodies, humanized antibodies that have been artificially modified in order to reduce heterologous antigenicity and the like against humans, can be appropriately produced if necessary for antibodies, such as mouse antibodies, mouse antibodies, rabbit antibodies , hamster antibodies, sheep antibodies and camel antibodies. Chimeric antibodies are antibodies composed of variable regions of the heavy chain and antibody light chain of a non-human mammal, such as mouse antibody, and constant regions of the human antibody heavy chain and light chain. They can be obtained by binding DNA that encodes a variable region of a mouse antibody to DNA that encodes a constant region of a human antibody, incorporating them into an expression vector, and introducing the vector into a host for producing the antibody. A humanized antibody, which is also called a reformed human antibody, can be synthesized by PCR of several oligonucleotides produced so that they have overlapping portions at the ends of DNA sequences designed to link the complementary determination regions (CDRs) of the antibody of a non-human mammal, such as a mouse. The DNA obtained can be linked to DNA encoding a human antibody constant region. Bound DNA can be incorporated into an expression vector, and the vector can be introduced into a host to produce the antibody (see EP239400 and WO96 / 02576). Human antibody FRs that are linked via CDR are selected when the CDR forms a favorable antigen binding site. If necessary, amino acids in the conserved region of an antibody variable region can be replaced such that the CDR of the reformed human antibody forms an appropriate antigen binding site (K. Sato et al., Cancer Res. 1993, 53: 851-856). The monoclonal antibodies of the present invention include such humanized antibodies and chimeric antibodies.
[0171] [000171] When the antibodies of the present invention are chimeric antibodies or humanized antibodies, the regions contained in these antibodies are preferably derived from human antibodies. For example, Cg1, Cg2, Cg3, and Cg4 can be used for the heavy chain, while Ck and Cl can be used for the light chain. In addition, the human antibody constant region can be modified as needed to improve the antibody or its production stability. A chimeric antibody of the present invention preferably comprises a variable region of an antibody derived from a non-human mammal and a constant region of a human antibody. However, a humanized antibody of the present invention preferably comprises CDRs of an antibody derived from a non-human mammal, and FRs and C regions of the human antibody. The human antibody derived constant regions comprise specific amino acid sequences, which vary depending on the isotype, such as IgG (IgG1, IgG2, IgG3, and IgG4), IgM, IgA, IgD and IgE. The constant regions used to prepare the humanized antibodies of the present invention can be antibody constant regions of any isotype. A human IgG constant region is preferably used, but the constant regions are not limited to that. However, there are no particular limitations on FRs derived from the human antibody that are used to prepare humanized antibodies, and can be derived from an antibody of any isotype.
[0172] [000172] The variable and constant regions of chimeric or humanized antibodies of the present invention can be modified by deletion, substitution, insertion and / or addition, while the antibodies exhibit the same binding specificity as the original antibodies.
[0173] [000173] Chimeric and humanized antibodies using human-derived sequences are expected to be useful when administered to humans for therapeutic or similar purposes, since their antigenicity in the human body has been attenuated.
[0174] [000174] In the present invention, amino acids can be modified to alter the biological properties of an antibody.
[0175] [000175] Minibodies (low molecular weight antibodies) are useful as antibodies because of their in vivo kinetic properties and low cost production using E. coli, plant cells, or the like.
[0176] [000176] Antibody fragments are a type of minibody. Minibodies include antibodies that comprise an antibody fragment as its partial structure. The minibodies of the present invention are not particularly limited by their structure or method of production, although they are capable of binding to the antigen. Some minibodies have greater activity than that of an entire antibody (Orita et al., Blood (2005) 105: 562-566). In this application, "antibody fragments" are not particularly limited although they are a portion of an entire antibody (for example, entire IgG). However, the antibody fragments preferably comprise a variable region of the heavy chain (VH) or a variable region of the light chain (VL). Preferred antibody fragments include, for example, Fab, F (ab ') 2, Fab' and Fv. The amino acid sequence of a variable region of the heavy chain (VH) or variable region of the light chain (VL) in an antibody fragment can be modified by substitution, deletion, addition and / or insertion. In addition, some portions of a variable region of the heavy chain (VH) or variable region of the light chain (VL) can be deleted, although the fragments retain their ability to bind to the antigen. For example, of the above antibody fragments, "Fv" is a minimal antibody fragment that comprises complete antigen recognition and binding sites. "Fv" is a dimer (VH-VL dimer) in which the variable region of the heavy chain (VH) and a variable region of the light chain (VL) are firmly linked by non-covalent bonding. The three complementarity determining regions (CDRs) of each variable region form an antigen-binding site on the surface of the VH-VL dimer. Six CDRs provide an antigen-binding site to the antibody. However, even a variable region (or half of an Fv comprising only three antigen-specific CDRs) has the ability to recognize and bind to an antigen, although its affinity is lower than that of the complete binding site. Accordingly, such molecules that are smaller than Fv are also included in the antibody fragments of the present invention. In addition, the variable regions of an antibody fragment can be chimerized or humanized.
[0177] [000177] It is preferable that the minibodies comprise both a variable region of the heavy chain (VH) and a variable region of the light chain (VL). Minibodies include, for example, antibody fragments, such as Fab, Fab ', F (ab') 2, and Fv, and scFv (single chain Fv) that can be prepared using antibody fragments (Huston et al., Proc. Natl. Acad. Sci. USA (1988) 85: 5879-83; Plickthun "The Pharmacology of Monoclonal Antibodies" Vol. 113, Resenburg and Moore (eds.), Springer Verlag, New York, pp. 269-315, (1994)); diabody (Holliger et al., Proc. Natl. Acad. Sci. USA (1993) 90: 6444-8; EP 404097; WO93 / 11161; Johnson et al., Method in Enzymology (1991) 203: 88-98; Holliger et al., Protein Engineering (1996) 9: 299305; Perisic et al., Structure (1994) 2: 1217-26; John et al., Protein Engineering (1999) 12 (7): 597-604; Atwell et al ., Mol. Immunol. (1996) 33: 1301-12); sc (Fv) 2 (Hudson et al., J Immunol. Methods (1999) 231: 177-89; Orita et al., Blood (2005) 105: 562-566); triabodies (Journal of Immunological Methods (1999) 231: 177-89); and tandem bodies (Cancer Research (2000) 60: 4336-41).
[0178] [000178] An antibody fragment can be prepared by treating an antibody with an enzyme, for example, a protease, such as papain and pepsin (see Morimoto et al., J. Biochem. Biophys. Methods (1992) 24: 107 -17; Brennan et al., Science (1985) 229: 81). Alternatively, an antibody fragment can also be produced by genetic recombination based on its amino acid sequence.
[0179] [000179] A minibody comprising a structure that results from the modification of an antibody fragment can be constructed using an antibody fragment obtained by enzyme treatment or genetic recombination. Alternatively, after constructing a gene that encodes an entire minibody and introducing it into an expression vector, the minibody can be expressed in appropriate host cells (see, for example, Co et al., J. Immunol. (1994) 152: 296876 ; Better and Horwitz, Methods Enzymol. (1989) 178: 476-96; Pluckthun and Skerra, Methods Enzymol. (1989) 178: 497-515; Lamoyi, Methods Enzymol. (1986) 121: 652-63; Rousseaux et al ., Methods Enzymol. (1986) 121: 663-9; Bird and Walker, Trends Biotechnol. (1991) 9: 132-7).
[0180] [000180] The above scFv is a single chain polypeptide comprising two variable regions linked together through a linker or the like, as needed. Two variable regions contained in an scFv are typically a VH and a VL, but an scFv can have two VH or two VL. In general, scFv polypeptides comprise a linker between VH and VL domains, thereby forming a paired portion of VH and VL necessary for antigen binding. A peptide linker of ten or more amino acids is typically used as the linker between VH and VL to form an intramolecularly paired portion between VH and VL. However, the scFv linkers of the present invention are not limited to such peptide linkers, although they do not inhibit scFv formation. To review scFv, see Pluckthun "The Pharmacology of Monoclonal Antibody", Vol. 113 (Rosenburg and ed. Moore, Springer Verlag, NY, pp.269-315 (1994)).
[0181] [000181] However, "diabody (Db)" refers to divalent antibody fragments constructed by gene fusion (P. Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); EP 404,097; WO93 / 11161; etc.). Diabodies are dimers comprising two polypeptide chains, in which each polypeptide chain comprises within the same chain a variable region of the light chain (VL) and a variable region of the heavy chain (VH) linked via a rather short linker to prevent their interaction two domains, for example, a binder of approximately five residues. VL and VH encoded on the same polypeptide chain will form a dimer because the linker between VL and VH is too short to form the single chain V region fragment. Therefore, diabodies have two antigen-binding sites. In this case, when VL and VH directed against two different epitopes (a and b) are expressed simultaneously as combinations of VLa-VHb and VLb-VHa joined with a ligand of approximately five residues, they are secreted as bispecific Db.
[0182] [000182] Diabodies comprise two scFv molecules and thus have four variable regions. As a result, diabodies have two antigen-binding sites. Unlike situations in which scFv does not form dimers, in the formation of diabody, the length of the linker between VH and VL in each scFv molecule generally has approximately five amino acids when the linker is a peptide linker. However, the scFv ligand that forms a diabody is not limited to that peptide ligand, as long as it does not inhibit scFv expression and diabody formation.
[0183] [000183] Furthermore, it is preferred that the minibodies and antibody fragments of the present invention additionally comprise an amino acid sequence from a constant region of the antibody heavy chain and / or an amino acid sequence from a constant region of the light chain. Alteration of one or more nucleotides
[0184] [000184] In this application, "nucleotide alteration" means that gene manipulation or mutagenesis is performed to insert, delete or replace at least one nucleotide in a DNA so that the polypeptide encoded by DNA has amino acid residues of interest. Specifically, this means that the codon that encodes the original amino acid residue is replaced by a codon that encodes the amino acid residue of interest. Such nucleotide changes can be introduced using methods, such as site-directed mutagenesis (see, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488), PCR mutagenesis, and cassette mutagenesis. In general, mutant antibodies whose biological properties have been improved have shown an amino acid sequence homology and / or similarity of 70% or higher, more preferably 80% or more, and even more preferably 90% or more (for example, 95% or more, 97%, 98%, 99%, etc.), when compared to the amino acid sequence of the original antibody variable region. In this application, homology and / or sequence similarity are defined as the proportion of amino acid residues that are homologous (the same residue) or similar (amino acid residues classified in the same group based on the general properties of the amino acid side chains) to the residues of original amino acids, after maximizing the value of sequence homology by performing sequence alignment and introducing a gap as needed. In general, naturally occurring amino acid residues are classified into the following groups based on the characteristics of their side chains: (1) hydrophobic: alanine, isoleucine, valine, methionine and leucine; (2) hydrophilic neutrals: asparagine, glutamine, cysteine, threonine and serine; (3) acidic: aspartic acid and glutamic acid; (4) basic: arginine, histidine and lysine; (5) residues that have an influence on the conformation of chain: glycine and proline; and (6) aromatics: tyrosine, tryptophan and phenylalanine. The number of modified amino acids is, for example, ten, nine, eight, seven, six, five, four, three, two or one, but it is not limited to that.
[0185] [000185] In general, a total of six complementarity determining regions (CDRs; hypervariable regions) present in the variable regions of the heavy and light chain interact to form the antigen binding site (s) of an antibody. It is known that one of these variable regions has the ability to recognize and bind to the antigen, although the affinity is lower than when all binding sites are included. Accordingly, the polypeptides of the present invention having antigen binding activity can encode fragment portions containing the respective heavy chain and light chain antigen binding sites of the antibody while maintaining the desired antigen binding activity.
[0186] [000186] The methods of the present invention allow for the efficient preparation, for example, of desired polypeptide multimers that in fact have the activity described above.
[0187] [000187] In a preferred embodiment of the present invention, the appropriate amino acid residues to be "modified" can be selected from, for example, the amino acid sequences of variable regions of the heavy and light chain of the antibody and the sequences of amino acids from the variable region of the light chain and antibody light chain. DNA expression
[0188] [000188] DNAs encoding the modified polypeptides are cloned (inserted) into an appropriate vector and then introduced into host cells. There is no particular limitation of the vectors while stably inserting the inserted nucleic acids. For example, using E. coli as the host, the vectors include cloning vectors. Preferred cloning vectors include pBluescript (Stratagene) vectors. It is possible to use several commercially available vectors. Expression vectors are particularly useful as vectors for producing polypeptide or polypeptide multimers of the present invention. There is no particular limitation on expression vectors while expressing polypeptides in vitro, in E. coli, cultured cells, or organisms. Preferred vectors include, for example, pBEST (Promega) vectors for in vitro expression; pET (Invitrogen) vectors for expression in E. coli; the pME18S-FL3 vector (GenBank Accession No. AB009864) for expression in culture cells; and the vector pME18S (Mol. Cell. Biol. 8: 466-472 (1988)) for expression in organisms. DNAs can be inserted into vectors by conventional methods, such as ligase reaction using restriction enzyme sites (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.4-11.11).
[0189] [000189] There is no particular limitation of the host cells above, and several host cells can be used depending on the target. Cells to express polypeptides include, for example, bacterial cells (for example, Streptococcus, Staphylococcus, E. coli, Streptomyces and Bacillus subtilis), fungal cells (for example, yeast and Aspergillus), insect cells (for example, Drosophila S2 and Spodoptera SF9), animal cells (e.g., CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cell), and plant cells. Vectors can be introduced into host cells using known methods, such as the calcium phosphate precipitation method, electroporation method (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1 -9.9), lipofection method and microinjection method.
[0190] [000190] In order to secrete polypeptides expressed by the host cell in the lumen of the endoplasmic reticulum, periplasmic space, or extracellular environment, appropriate secretion signals can be incorporated into the polypeptides of interest. These signals may be intrinsic or foreign to the polypeptides of interest.
[0191] [000191] Expression vectors of the first, second, third and fourth polypeptides can be constructed by inserting DNAs that encode the polypeptides individually in separate vectors. Alternatively, some DNAs that encode the first, second, third and fourth polypeptide (for example, a DNA that encodes the first polypeptide and a DNA that encodes the second polypeptide) can be inserted into a single vector to construct expression vectors. When an expression vector is constructed by inserting multiple DNAs into a single vector, there is no limitation on the combination of DNAs encoding the polypeptide to be inserted. Recovery of expression products
[0192] [000192] When the polypeptides are secreted into a culture medium, the expression products are recovered by collecting the medium. When polypeptides are produced in cells, cells are first lysed and then polypeptides are collected.
[0193] [000193] Polypeptides can be collected and purified from a recombinant cell culture by known methods including precipitation in ammonium sulfate or ethanol, acid extraction, anionic or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography , hydroxylapatite chromatography and lectin chromatography.
[0194] [000194] Protein A affinity chromatography is preferably used in the present invention.
[0195] [000195] Protein A columns include, but are not limited to, Hyper D (PALL), POROS (Applied Biosystems), Sepharose F.F. (GE), and ProSep (Millipore). Alternatively, protein A affinity chromatography can be performed using a resin bound by a ligand that mimics the IgG binding capacity of protein A. Also when such protein A mimetic is used, the polypeptide multimers of interest can be isolated and purified creating a difference in binding capacity as a result of the amino acid modifications of the present invention. Such protein A mimetics include, but are not limited to, for example, mabSelect SuRE (GE Healthcare).
[0196] [000196] Furthermore, the present invention provides polypeptide multimers obtained by the methods of purification or production of the present invention.
[0197] [000197] The present invention also provides polypeptide multimers comprising the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or without antigen binding activity, wherein protein A binding capacity is different between the first and second polypeptide.
[0198] [000198] Such polypeptide multimers can be obtained by the methods described in this application. The structures and properties of the polypeptide multimers are as described above, and summarized below.
[0199] [000199] When compared to before the amino acid modification, the protein A binding capacity of the polypeptide multimers of the present invention has been altered. More specifically, protein A binding capacity has been altered in one or both of the first polypeptide having antigen binding activity and in the second polypeptide having antigen binding activity or without antigen binding activity. In a polypeptide multimer of the present invention, the protein A binding capacity of the first polypeptide having antigen binding activity is different from that of the second polypeptide having antigen binding activity or without antigen binding activity. Consequently, the pH of solvent for elution in protein A is different for the first polypeptide and the second polypeptide in affinity chromatography.
[0200] [000200] In addition, the first polypeptide and / or the second polypeptide can form a multimer with one or two third polypeptides.
[0201] [000201] Thus, the present invention relates to polypeptide multimers comprising the first polypeptide having an antigen binding activity, the second polypeptide having antigen binding activity or without antigen binding activity, and one or two third polypeptides having antigen-binding activity, in which protein A binding capacity is different for the first and second polypeptides. Such polypeptide multimers can also be obtained by the methods described in this application.
[0202] [000202] Polypeptide multimers may additionally comprise a fourth polypeptide. Any of the first polypeptide and the second polypeptide can form a multimer with the third polypeptide, while the other can form another multimer with the fourth polypeptide.
[0203] [000203] Thus, the present invention relates to polypeptide multimers comprising the first polypeptide having antigen binding activity, the second polypeptide having antigen binding activity or without antigen binding activity, the third polypeptide having an antigen binding activity, and the fourth polypeptide having an antigen binding activity, in which protein A binding capacity is different for the first and second polypeptide. Such polypeptide multimers can also be obtained by the methods described in this application.
[0204] [000204] The first polypeptide above having antigen binding activity and second polypeptide having antigen binding activity or without antigen binding activity can comprise an amino acid sequence from a constant region of the antibody heavy chain or a sequence of amino acids of an antibody Fc domain. The amino acid sequence of an antibody heavy chain constant region or an antibody the Fc domain includes, but is not limited to, an amino acid sequence of a human IgG-derived constant region.
[0205] [000205] However, the third polypeptide above having antigen binding activity and the fourth polypeptide having antigen binding activity may comprise an amino acid sequence from a constant region of the antibody light chain.
[0206] [000206] In addition, polypeptides having antigen-binding activity can comprise an amino acid sequence from a variable region of antibody (e.g., the amino acid sequences of CDR1, CDR2, CDR3, FR1, FR2, FR3 and FR4) .
[0207] [000207] The first polypeptide above having antigen binding activity and second polypeptide having antigen binding activity or without antigen binding activity can comprise an amino acid sequence of an antibody heavy chain, or amino acid sequence comprising a variable region of the antibody light chain and a constant region of the antibody heavy chain. The above third polypeptide having antigen-binding activity and fourth polypeptide having antigen-binding activity can comprise an amino acid sequence from an antibody light chain, or amino acid sequence that comprises a variable region of the antibody heavy chain and a constant region of the antibody light chain.
[0208] [000208] A polypeptide multimer of the present invention can be a multispecific antibody. The multispecific antibodies of the present invention include, but are not limited to, bispecific antibodies capable of specifically binding two types of antigens.
[0209] [000209] In a polypeptide multimer of the present invention, one or more amino acid residues have been modified so that there is (a greater) difference in protein A binding capacity between the first polypeptide having an antigen binding activity and the second polypeptide having antigen-binding activity or no antigen-binding activity. As described above, the modification sites include, but are not limited to, for example, the following amino acid residues: TLMISR at positions 250-255, VLHQDWLNGK at positions 308-317, EALHNHY at positions 430436, preferably TLMIS at positions 250-254 , LHQD at positions 309-312, LN at positions 314-315, E at position 430, LHNHY at positions 432-436, more preferably LMIS at positions 251-254, LHQ at positions 309-311, L at position 314, LHNH at positions 432-435, and particularly LMIS at positions 252-254, L at position 309, Q at position 311, and NHY at positions 434-436 (EU numbering) in an antibody Fc domain or a heavy chain constant region. However, for amino acid modifications of a variable region of the antibody heavy chain, the preferred sites of modification include FR1, CDR2 and FR3.
[0210] [000210] More specifically, the polypeptide multimers of the present invention include, but are not limited to, polypeptide multimers in which the amino acid residue at position 435 (EU numbering) in the amino acid sequence of an antibody Fc domain or constant region of the chain heavy antibody is histidine or arginine in one of the first polypeptide having antigen binding activity and the second polypeptide having antigen binding activity or without antigen binding activity, while another polypeptide has a different amino acid residue at position 435 (EU numbering) in the amino acid sequence of an antibody Fc domain or constant region of the antibody heavy chain.
[0211] [000211] In addition, the polypeptide multimers of the present invention include, but are not limited to, polypeptide multimers in which the amino acid residue at position 435 (EU numbering) in the amino acid sequence of an antibody constant region heavy chain is histidine in one of the first polypeptide having an antigen-binding activity and the second polypeptide having an antigen-binding activity or without antigen-binding activity, while the amino acid residue at position 435 (EU numbering) in the amino acid sequence of a chain heavy antibody constant region is arginine in another polypeptide.
[0212] (1) Multímeros polipeptídicos que compreendem o primeiro ou segundo polipeptídeo compreendendo uma sequência de aminoácidos na qual os resíduos de aminoácidos nas posições 435 e 436 (numeração EU) na sequência de aminoácidos de uma região constante da cadeia pesada de anticorpo derivada de uma IgG humana foram modificados para histidina (His) e tirosina (Tyr), respectivamente. [000212] In addition, the polypeptide multimers of the present invention comprising the first and second polypeptides include, but are not limited to, the examples below. (1) Polypeptide multimers comprising the first or second polypeptide comprising an amino acid sequence in which the amino acid residues at positions 435 and 436 (EU numbering) in the amino acid sequence of an antibody heavy chain region derived from a human IgG were changed to histidine (His) and tyrosine (Tyr), respectively.
[0213] [000213] Such polypeptide multimers include, but are not limited to, for example, polypeptide multimers comprising the first or second polypeptide comprising the amino acid sequence of SEQ ID NO: 9, 11, 13 or 15. (2) Polypeptide multimers comprising the first or second polypeptide comprising an amino acid sequence in which the amino acid residues at positions 435 and 436 (EU numbering) in the amino acid sequence of an antibody heavy chain region derived from a human IgG were modified to arginine (Arg) and phenylalanine (Phe), respectively.
[0214] [000214] Such polypeptide multimers include, but are not limited to, for example, polypeptide multimers comprising the first or second polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or 12. (3) Polypeptide multimers comprising the first or second polypeptide comprising an amino acid sequence in which the amino acid residues at positions 435 and 436 (EU numbering) in the amino acid sequence of an IgG-derived antibody heavy chain region were modified to arginine (Arg) and tyrosine (Tyr), respectively.
[0215] [000215] Such polypeptide multimers include, but are not limited to, for example, polypeptide multimers comprising the first or second polypeptide comprising the amino acid sequence of SEQ ID NO: 14. (4) Polypeptide multimers comprising the first and second polypeptides, wherein any of the polypeptides comprises an amino acid sequence in which the amino acid residues at positions 435 and 436 (EU numbering) in the amino acid sequence of a constant region of the heavy chain of antibody derived from a human IgG were modified to histidine (His) and tyrosine (Tyr), respectively; and another polypeptide comprises an amino acid sequence in which the amino acid residues at positions 435 and 436 (EU numbering) in the amino acid sequence of a constant region of the antibody heavy chain have been modified to arginine (Arg) and phenylalanine (Phe), respectively .
[0216] [000216] Such polypeptide multimers include, but are not limited to, for example, polypeptide multimers comprising the first polypeptide comprising the amino acid sequence of SEQ ID NO: 9, 11, 13 or 15 and the second polypeptide comprising the sequence of amino acids of SEQ ID NO: 10 or 12. (5) Polypeptide multimers comprising the first and second polypeptides, wherein any of the polypeptides comprises an amino acid sequence in which the amino acid residues at positions 435 and 436 (EU numbering) in the amino acid sequence of a constant region of the heavy chain of antibody derived from a human IgG were modified to histidine (His) and tyrosine (Tyr), respectively; and another polypeptide comprises an amino acid sequence in which the amino acid residues at positions 435 and 436 (EU numbering) in the amino acid sequence of a constant region of the antibody heavy chain have been modified to arginine (Arg) and tyrosine (Tyr), respectively .
[0217] [000217] Such polypeptide multimers include, but are not limited to, for example, polypeptide multimers comprising the first polypeptide comprising the amino acid sequence of SEQ ID NO: 9, 11, 13 or 15 and the second polypeptide comprising the amino acid sequence SEQ ID NO: 14. (6) Polypeptide multimers comprising the first and second polypeptides, wherein any of the polypeptides comprises an amino acid sequence in which the amino acid residues at positions 435 and 436 (EU numbering) in the amino acid sequence of a constant region of the heavy chain of antibody derived from a human IgG were modified to arginine (Arg) and phenylalanine (Phe), respectively; and another polypeptide comprises an amino acid sequence in which the amino acid residues at positions 435 and 436 (EU numbering) in the amino acid sequence of a constant region of the antibody heavy chain have been modified to arginine (Arg) and tyrosine (Tyr), respectively .
[0218] [000218] Such polypeptide multimers include, but are not limited to, for example, polypeptide multimers comprising the first polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or 12 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 14.
[0219] [000219] The first and second polypeptide above may additionally comprise a variable region of the antibody heavy chain. The polypeptide multimers of (1) to (6) above may also comprise the third polypeptide and / or the fourth polypeptide.
[0220] [000220] In addition, the present invention provides polypeptide variants comprising a polypeptide comprising a mutation in the amino acid residue at position 435 or 436 (EU numbering). Such polypeptide variants include, but are not limited to, polypeptide variants comprising a polypeptide described in the Examples.
[0221] [000221] In addition, the present invention provides nucleic acids that encode a polypeptide (polypeptide having antigen binding activity) that constitutes a polypeptide multimer of the present invention. The present invention also provides vectors that carry such nucleic acids.
[0222] [000222] The present invention also provides host cells that comprise the above nucleic acids or vectors. There is no particular limitation on host cells, and include, for example, E. coli and various plant and animal cells. Host cells can be used, for example, as a production system to produce and express the polypeptide or polypeptide multimers of the present invention. There are in vitro and in vivo production systems to produce the polypeptide or polypeptide multimers. In vitro production systems include those using eukaryotic cells and prokaryotic cells.
[0223] [000223] Eukaryotic cells that can be used as host cells include, for example, animal cells, plant cells and fungal cells. Animal cells include: mammalian cells, for example, CHO (J. Exp. Med. (1995) 108, 945), COS, HEK293, 3T3, myeloma, BHK (hamster puppy kidney), HeLa, and Vero; amphibious cells, such as Xenopus laevis oocytes (Valle, et al., Nature (1981) 291: 338-340); and insect cells, such as Sf9, Sf21, and Tn5. To express the polypeptide or polypeptide multimers of the present invention, CHO-DG44, CHO-DX11B, COS7 cells, HEK293 cells and BHK cells can be used appropriately. Of the animal cells, CHO cells are particularly preferred for large-scale expression. Vectors can be introduced into a host cell, for example, by calcium phosphate methods, DEAE-dextran methods, methods using DOTAP cationic liposome (Boehringer-Mannheim), electroporation methods or lipofection methods.
[0224] [000224] It is known that plant cells, such as cells derived from Nicotiana tabacum and cells of Lemna minor are protein production systems, and these cells can be used to produce polypeptide multimers or polypeptides of the present invention by methods that cultivate calluses from these cells. Protein expression systems that use fungal cells including yeast cells, for example, cells of the genus Saccharomyces (Saccharomyces cerevisiae, Saccharomyces pombe, etc.), and cells of filamentous fungi, for example, the genus Aspergillus (Aspergillus niger, etc. ) are known, and these cells can be used as a host to produce polypeptide multimers or polypeptides of the present invention.
[0225] [000225] When prokaryotic cells are used, production systems that use bacterial cells are available. Production systems that use bacterial cells including Bacillus subtilis as well as E. coli described above are known, and can be used to produce polypeptide multimers or polypeptides of the present invention.
[0226] [000226] When a polypeptide multimer or polypeptide is produced using a host cell of the present invention, a polynucleotide encoding the polypeptide multimer or polypeptide of the present invention can be expressed by culturing the transformed host cell with an expression vector comprising the polynucleotide. Culture can be carried out according to known methods. For example, when animal cells are used as a host, DMEM, MEM, RPMI 1640, or IMDM can be used as a culture medium. Culture medium can be used with serum supplement solutions, such as FBS or fetal calf serum (FCS). Alternatively, the cells can be grown in cultures without serum. The preferred pH is approximately 6 to 8 during the course of the culture. Incubation is typically performed at approximately 30 to 40 ° C for approximately 15 to 200 hours. Medium is changed, ventilated, or agitated, as needed.
[0227] [000227] On the other hand, systems for producing polypeptides in vivo include, for example, those using animals and those using plants. A polynucleotide of interest is introduced into an animal or plant to produce the polypeptide in the body of the animal or plant, and then the polypeptide is collected. The "host" of the present invention includes such animals and plants.
[0228] [000228] When animals are used, production systems that use mammals or insects are available. Mammals, such as goat, pig, sheep, mouse and cattle can be used (Vicki Glaser, SPECTRUM Biotechnology Applications (1993)). When mammals are used, transgenic animals can be used.
[0229] [000229] For example, a polynucleotide encoding a polypeptide or polypeptide multimer of the present invention can be prepared as a fusion gene with a gene encoding a polypeptide specifically produced in milk, such as goat β-casein. Next, fragments of polynucleotides containing this fusion gene are injected into goat embryos, which are then introduced back into goats. The antibody of interest can be obtained from the milk produced by the transgenic goats, which are born from the goats that received the embryos, or by their offspring. Appropriate hormones can be administered to transgenic goats to increase the volume of milk containing the antibody produced by transgenic goats (Ebert et al., Bio / Technology (1994) 12: 699-702).
[0230] [000230] Insects, such as silkworms, can be used to produce polypeptide or polypeptide multimers of the present invention. When silkworms are used, baculoviruses that carry a polynucleotide that encodes a polypeptide multimer or polypeptide of interest can be used to infect silkworms, so that the polypeptide multimer or polypeptide of interest can be obtained from the body fluids of these silkworms ( Susumu et al., Nature (1985) 315: 592-594).
[0231] [000231] Plants used to produce polypeptide multimers or polypeptides of the present invention include, for example, tobacco. When tobacco is used, a polynucleotide that encodes a polypeptide multimer or polypeptide of interest is inserted into a plant expression vector, for example, pMON 530, and then the vector is introduced into a bacterium, such as Agrobacterium tumefaciens. The bacteria are then used to infect tobacco, such as Nicotiana tabacum, and the desired polypeptide or polypeptide multimer can be obtained from tobacco leaves (Ma et al., Eur. J. Immunol. (1994) 24: 131-138) . Alternatively, the same bacteria can be used to infect Lemna minor, and after cloning, the desired polypeptide or polypeptide multimer can be obtained from Lemna minor cells (Cox KM et al., Nat. Biotechnol. 2006 Dec; 24 (12): 1591-1597).
[0232] [000232] The polypeptide or polypeptide multimer obtained in this way can be isolated from the inside or outside (such as the medium and milk) of host cells, and purified as a substantially pure and homogeneous polypeptide multimer or polypeptide. The methods used to separate and purify a polypeptide or polypeptide multimer are not limited, and the methods used in the purification of standard polypeptide can be applied. Antibodies can be isolated and purified by selecting an appropriate combination, for example, of chromatographic columns, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis , recrystallization and the like.
[0233] [000233] Chromatographies include, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R Marshak et al., (1996) Cold Spring Harbor Laboratory Press). These chromatographies can be performed using liquid phase chromatography, such as HPLC and FPLC. Examples of affinity chromatography columns include protein A columns and protein G columns. Examples of columns using protein A include, but are not limited to, Hyper D, POROS, and Sepharose F. F. (Pharmacia).
[0234] [000234] As needed, modifications can be added and peptides can be deleted from a polypeptide multimer or polypeptide arbitrarily by treatment with an appropriate protein-modifying enzyme before or after purification of the polypeptide multimer or polypeptide. Such protein-modifying enzymes include, for example, trypsin, chymotrypsin, lysyl endopeptidase, protein kinase and glucosidase.
[0235] [000235] Another preferred embodiment of the present invention includes a method for producing a polypeptide or polypeptide multimer of the present invention, which comprises the steps of culturing the host cells of the present invention as described above and collecting the polypeptide from the cell culture.
[0236] [000236] In addition, the present invention relates to pharmaceutical compositions (agents) comprising a polypeptide or polypeptide multimer of the present invention and a pharmaceutically acceptable carrier. In the present invention, "pharmaceutical compositions" generally refer to agents for treatment or prevention, or testing and diagnosing diseases.
[0237] [000237] The pharmaceutical compositions of the present invention can be formulated by methods known to those skilled in the art. For example, such pharmaceutical compositions can be used parenterally in the form of injections, which are sterile solutions or suspensions prepared with water or other pharmaceutically acceptable liquid. For example, such compositions can be formulated by appropriately combining with a pharmaceutically acceptable carrier or medium, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, or the like, and mixed in a unit dose form that meets the generally accepted requirements for preparing pharmaceutical products. In such preparations, the amount of the active ingredient is adjusted such that a suitable amount within a specified range is obtained.
[0238] [000238] Sterile compositions for injection can be formulated using vehicles, such as distilled water for injection, according to standard formulation protocols.
[0239] [000239] Aqueous solutions for injection include, for example, physiological saline and isotonic solutions containing glucose or other adjuvants (for example, D-sorbitol, D-mannose, D-mannitol and sodium chloride). Suitable solubilizers, for example, alcohols (ethanol and the like), polyalcohols (propylene glycol, polyethylene glycol and the like), non-ionic surfactants (polysorbate 80TM, HCO-50 and the like) can be used in combination.
[0240] [000240] Oils include soy and sesame oils. Benzyl benzoate and / or benzyl alcohol can be used as solubilizers in combination. Buffers (for example, phosphate buffer and sodium acetate buffer), soothing agents (for example, procaine hydrochloride), stabilizers (for example, benzyl alcohol and phenol) and / or antioxidants can also be combined. Prepared injections are usually filled in appropriate ampoules.
[0241] [000241] The pharmaceutical compositions of the present invention are preferably administered parenterally. For example, the compositions can be in the form of injections, transnasal agents, transpulmonary agents, or transdermal agents. For example, such compositions can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection or the like.
[0242] [000242] The methods of administration can be appropriately selected considering the age and symptoms of a patient. The dosage of a pharmaceutical composition comprising a polypeptide or polypeptide multimer or a polynucleotide encoding a polypeptide or polypeptide multimer can be established, for example, within the range of 0.0001 to 1000 mg / kg in weight for each administration. Alternatively, the dosage can be, for example, from 0.001 to 100,000 mg per patient. However, in the present invention, the dosage is not necessarily limited to the ranges described above. Although the dosage and method of administration will vary depending on a patient's weight, age, symptoms, and the like, those skilled in the art can select the appropriate dosage and methods of administration considering these factors.
[0243] [000243] The multispecific antibodies of the present invention can be formulated by combining them with other pharmaceutical components as needed.
[0244] [000244] All prior art references cited in this application are incorporated by reference in this specification. EXAMPLES
[0245] [000245] In the following, the present invention will be specifically described with reference to the Examples, but should not be construed as limited to that. [Example 1] Construction of antibody gene expression vectors and expression of respective antibodies
[0246] [000246] The antibody H chain variable regions used were:
[0247] [000247] Q153 (the H chain variable region of an anti-human F.IX antibody, SEQ ID NO: 1), Q407 (the H chain variable region of an anti-human F.IX antibody, SEQ ID NO: 2), J142 (the H chain variable region of an anti-human FX antibody, SEQ ID NO: 3), J300 (the H chain variable region of an anti-human FX antibody, SEQ ID NO: 4), and MRA-VH (the H chain variable region of an anti-human interleukin-6 antibody, SEQ ID NO: 5).
[0248] [000248] The antibody L variable chain regions used were:
[0249] [000249] L180-k (an L chain common to an anti-human F.IX antibody and an anti-human FX antibody, SEQ ID NO: 6), L210-k (an L chain common to an anti-F.IX antibody) -human / anti-human FX antibody, SEQ ID NO: 7), and MRA-k (the L chain of an anti-human interleukin-6 antibody, SEQ ID NO: 8).
[0250] [000250] The antibody H chain constant regions used were:
[0251] [000251] G4d (SEQ ID NO: 9), which was constructed from IgG4 introducing a Pro substitution mutation for Ser at position 228 (EU numbering) and elimination of Gly and Lys C-terminals; z72 (SEQ ID NO: 10), which was constructed from G4d introducing the following mutations: a mutation replacing Arg with His at position 435 (EU numbering); a Tyr substitution mutation of Phe at position 436 (EU numbering); and a replacement mutation from Pro to Leu at position 445 (EU numbering); z7 (SEQ ID NO: 11), which was constructed from G4d introducing a Lys substitution mutation for Glu at position 356 (EU numbering); z73 (SEQ ID NO: 12), which was constructed from z72 introducing a Lys substitution mutation from Glu at position 439 (EU numbering); z106 (SEQ ID NO: 13), which was constructed from z7 introducing the following mutations: a Gln substitution mutation for Lys at position 196 (EU numbering); a Phe Tyr substitution mutation at position 296 (EU numbering); and a Lys substitution mutation for Arg at position 409 (EU numbering); z107 (SEQ ID NO: 14), which was constructed from z73 introducing the following mutations: a mutation replacing Gln with Lys at position 196 (EU numbering); a Phe Tyr substitution mutation at position 296 (EU numbering); a substitution mutation of Lys for Arg at position 409 (EU numbering); and a Phe Tyr substitution mutation at position 436 (EU numbering); and G1d (SEQ ID NO: 15), which was constructed by deleting Gly and Lys C-terminals from IgG1. Mutations of substitution of Lys for Glu at position 356 (EU numbering) and Glu for Lys at position 439 (EU numbering) were introduced for the efficient formation of heteromeric molecules of the respective H chains in the production of heteromeric antibodies ((WO 2006/106905 ) PROCESS FOR PRODUCTION OF POLYPEPTIDE BY REGULATION OF ASSEMBLY).
[0252] [000252] The Q153-G4d and Q153-z7 genes of the anti-human F.IX antibody H chain were constructed by linking respectively G4d and z7 downstream of Q153. The anti-human F.IX antibody H chain Q407-z106 gene was constructed by ligating z106 downstream of Q407. The J142-G4d, J142-z72 and J142-z73 genes of the anti-human F.X antibody H chain were constructed by linking G4d, z72, and z73 respectively downstream of J142. The anti-human F.X antibody H chain J300-z107 gene was constructed by ligating z107 downstream of J300. The MRA-G1d, MRA-z106 and MRA-z107A genes of the H chain of the human interleukin-6 anti-receptor antibody were constructed by linking G1d, z106, and z107 respectively downstream of MRA-VH.
[0253] [000253] The respective antibody genes (Q153-G4d, Q153-z7, Q407-z106, J142-G4d, J142-z72, J142-z73, J300-z106, MRA-G1d, MRA-z106, MRA-z107, L180 -k, L210-k and MRA-k) were inserted into animal cell expression vectors.
[0254] [000254] The following antibodies were expressed transiently in FreeStyle293 cells (Invitrogen) by transfection using the constructed expression vectors. As shown below, the antibodies were named using the transfected antibody gene combinations. MRA-G1d / MRA-k MRA-z106 / MRA-z107 / MRA-k Q153-G4d / J142-G4d / L180-k Q153-G4d / J142-z72 / L180-k Q153-z7 / J 142-z73 / L180-k Q407-z106 / J300-z107 / L210-k [Example 2] Evaluation of elution conditions for protein A affinity chromatography
[0255] [000255] Q153-G4d / J142-G4d / L180-k and Q153-G4d / J142-z72 / L180-k were transiently expressed, and the resulting FreeStyle293 cell culture medium (hereinafter abbreviated as CM) was used as a sample to evaluate the elution conditions of protein A affinity chromatography. The CM samples were filtered through a 0.22 μm pore size filter, and loaded onto a rProtein A Sepharose Fast Flow (GE Healthcare) column equilibrated with D-PBS. The column was subjected to washes 1 and 2 and elutions 1 to 5 in a gradual manner as shown in Table 1. The volume of CM to be loaded into the column was adjusted to 20 mg antibody / ml of resin. The fractions eluted under each condition were collected, and the respective eluted fractions were analyzed by cation exchange chromatography to identify their components. To prepare controls, each CM was loaded with rProtein G Sepharose Fast Flow resin (GE Healthcare). Samples purified by batch elution were used as controls. Since the G protein binds to the Fab domain of an antibody, all antibody species (a bispecific antibody of interest in which the two types of H chains associate in a heteromeric manner (heteromeric antibody) and as an impurity , monospecific homomeric antibodies in which single-type H chains are homomerically associated) in CM can be purified using protein G, despite its binding affinity for protein A. Table 1
[0256] [000256] The CM in which Q153-G4d / J142-G4d / L180-k or Q153-G4d / J142-z72 / L180-k had been expressed was eluted from a protein A column (elution 1 to 5), and the respective eluted fractions were analyzed by cation exchange chromatography. As for Q153-G4d / J142-G4d / L180-k, the analysis revealed that as the elution condition was changed from 1 to 5, that is, as the pH of the elution buffer was reduced, the antibody composition of the eluted fractions gradually changed in the order of the homomeric antibody J142-G4d / L180-k to the heteromeric antibody Q153-G4d / J142-G4d / L180-k, and then to the homomeric antibody Q153-G4d / L180-k. It is understood that the order of elution depends on the binding capacity of protein A. This implies that the homomeric antibody Q153-G4d / L180-k, which remained bound until exposed to low pH, has a greater binding capacity for protein A than the homomeric species J142-G4d / L180-k (a homomeric antibody against FX) eluted at a high pH. The variable region J142 is known to be a sequence incapable of binding to protein A. Specifically, the homomeric species J142-G4d / L180-k (a homomeric antibody against FX) has two protein A binding sites; the heteromeric antibody Q153-G4d / J142-G4d / L180-k has three; and the homomeric antibody Q153-G4d / L180-k (homomeric antibody against FX) has four protein A binding sites. Thus, it was revealed that more protein A binding sites resulted in stronger protein A binding, and therefore a lower pH was required for elution.
[0257] [000257] However, as for Q153-G4d / J142-z72 / L180-k, it was revealed that as the elution condition was changed from 1 to 5, the antibody composition in the modified eluted fraction of the heteromeric antibody Q153-G4d / J142 -z72 / L180-k to the homomeric antibody Q153-G4d / L180-k. The homomeric antibody J142-z72 / L180-k (a homomeric antibody against FX) was barely detectable in any eluted fraction. This suggests that J142-z72 / L180-k has no protein A binding capacity. It is believed that the lack of protein A binding capacity of J142-z72 could be due to the substitution mutation introduced from Arg to His in the 435 (EU numbering). The homomeric antibody J142-z72 / L180-k (a homomeric antibody against FX) has no protein A binding site, while the heteromeric antibody Q153-G4d / J142-z72 / L180-k has two protein A binding sites and the homomeric antibody Q153-G4d / L180-k (a homomeric antibody against FIX) has four. The homomeric antibody J142-z72 / L180-k (a homomeric antibody against FX) passes through the column because it does not bind to protein A. This is the reason why J142-z72 / L180-k was undetectable in any eluted fraction. In addition, in both cases of Q153-G4d / J142-G4d / L180-k and Q153-G4d / J142-z72 / L180-k, it has been suggested that the heteromeric antibody and the homomeric antibody Q153-G4d / L180-k ( homomeric antibody against FIX) were separable from one another at pH 3.6 or a lower pH. [Example 3] Isolation and purification of heteromeric antibodies by protein A chromatography
[0258] [000258] CM samples containing the following antibodies were used: Q153-G4d / J142-G4d / L180-k Q153-G4d / J142-z72 / L180-k Q153-z7 / J 142-z73 / L180-k Q407-z106 / J300-z107 / L210-k
[0259] [000259] The CM samples were filtered through a filter with a pore size of 0.22 μm, and loaded onto a rProtein A Sepharose Fast Flow column (GE Healthcare) equilibrated with D-PBS. The column was subjected to washes 1 and 2 and elutions 1 and 2 as shown in Table 2 (except that Q407-z106 / J300-z107 / L210-k was subjected to elution 1 only). The elution conditions were determined based on the result described in Example 2. The volume of the CM to be loaded onto the column was adjusted to 20 mg antibody / ml of resin. The respective fractions eluted under each condition were collected and analyzed by cation exchange chromatography to identify their components. To prepare the controls, each CM was loaded on rProtein G Sepharose Fast Flow resin (GE Healthcare) in the same manner as described in Example 2. Samples purified by batch elution were used as controls. Table 2
[0260] [000260] The result of the cation exchange chromatography analysis of each eluted fraction is shown in Table 3 below. The values represent the peak elution area expressed as a percentage. Except for the Q153-G4d / J142-G4d / L180-k antibody, homomeric antibodies against FX were barely detectable in any eluted fraction. Thus, it was revealed that not only the homomeric antibody J142-z72 (a homomeric antibody against FX) described in Example 2 but also the homomeric antibodies J142-z73 and J300-z107 (a homomeric antibody against FX) were unable to bind to the protein A. It is believed that the lack of protein A binding capacity in the homomeric antibody against FX was due to the Arg to His substitution mutation at position 435 (EU numbering), which was introduced into the H chain constant region of the antibody against FX. The heteromeric antibody, which is a bispecific antibody of interest, was detected mostly in the elution fraction 1. However, most homomeric antibodies against FIX were eluted by elution 2, although they were also detected at a very low level in the fraction of elution. elution 1. When compared to Q153-G4d / J142-z72 / L180-k, in the case of Q153-z7 / J142-z73 / L180-k and Q407-z106 / J300-z107 / L210-k, the proportion of the heteromeric antibody (the bispecific antibody of interest) was considerably increased in the fraction eluted at pH 3.6. Thus, it was demonstrated that when the substitution mutations of Lys by Glu at position 356 (EU numbering) and Glu by Lys at position 439 (EU numbering) for the efficient formation of heteromeric molecules of the respective H chains were introduced in combination with the Arg to His substitution mutation at position 435 (EU numbering), the heteromeric antibody (bispecific antibody of interest) can be purified to a purity of 98% or more by the protein A-based purification step alone.
[0261] [000261] As described above, the present inventors have revealed that based on differences in the number of protein A binding sites between the heteromeric antibody and homomeric antibodies, the heteromeric antibody can be isolated and purified to high purity by the protein A chromatography step by herself. Table 3
[0262] [000262] As described in Example 3 above, the present inventors have demonstrated that by using z106 (SEQ ID NO: 13) and z107 (SEQ ID NO: 14) for the respective H chain regions of the bispecific antibody, the heteromeric antibody (antibody bispecific of interest) can be purified to a purity of 98% or more by the protein A step alone. However, loss of protein A binding affinity probably results in loss of human FcRn binding activity because protein A and human FcRn recognize the same site in an IgG antibody (J Immunol. 2000, 164 (10): 5313- 8). In fact, there is a reported method for purifying a bispecific antibody to 95% purity using protein A. The method uses a mouse IgG2b H chain that does not bind to protein A. Catumaxomab (a bispecific antibody) purified by this method has a half-life of approximately 2.1 days in humans. Its half-life is significantly shorter than the half-life of a normal human IgG1 that is 2 to 3 weeks (Non-Patent Document 2). In this context, antibodies having z106 (SEQ ID NO: 13) and z107 (SEQ ID NO: 14) described in Example 3 as constant regions were evaluated for their pharmacokinetics.
[0263] [000263] In a pharmacokinetic experiment to calculate half-life in humans, the pharmacokinetics in transgenic mice with human FcRn (mice B6.mFcRn - / -. HFcRn strain Tg 276 + / +, Jackson Laboratories) was evaluated by the following procedure . MRA-G1d / MRA-k (hereinafter abbreviated as MRA-IgG1) having the IgG1 and MRA-z106 / MRA-z107 / MRA-k constant region (hereinafter abbreviated MRA-z106 / z107) with z106 / z107 as constant region was each administered intravenously once at a dose of 1 mg / kg to mice, and blood was collected at appropriate time points. The collected blood was immediately centrifuged at 15,000 rpm and 4 ° C for 15 minutes to obtain blood plasma. The separated plasma was stored in a refrigerator at -20 ° C or below until use. The plasma concentration was determined by ELISA.
[0264] [000264] MRA-IgG1 and MRA-z106 / z107k were evaluated for their plasma retention in transgenic mice with human FcRn. As shown in Fig. 1, the result indicates that the retention of MRA-z106 / z107 in the plasma was comparable or longer than that of MRA-IgG1. As described above, z106 / z107, a constant region that allows efficient production or purification of heteromeric antibody at high purity by the protein A-based purification step alone, has been shown to be comparable or superior to human IgG1 in terms of plasma retention. [Example 5] Construction of antibody gene expression vectors and expression of the respective antibodies
[0265] [000265] The antibody H chain variable regions used were: Q499 (the H chain variable region of an anti-human F.IX antibody, SEQ ID NO: 16). J339 (the H chain variable region of an anti-human FX antibody, SEQ ID NO: 17).
[0266] [000266] The antibody L chain used was: L377-k (the L chain common to an anti-human F.IX antibody and an anti-human FX antibody, SEQ ID NO: 18).
[0267] [000267] The antibody H chain constant regions used were: z118 (SEQ ID NO: 19), which was constructed from z106 described in Example 1, introducing a Leu substitution mutation of Phe at position 405 (EU numbering); z121 (SEQ ID NO: 20), which was constructed from z118 by introducing an Arg to His substitution mutation at position 435 (EU numbering); and z119 (SEQ ID NO: 21), which was constructed from z118 by introducing Glu substitution mutations for Lys at position 356 (EU numbering) and Lys for Glu at position 439 (EU numbering).
[0268] [000268] The Q499-z118 and Q499-z121 genes of the anti-human F.IX antibody H chain were constructed by linking z118 and z121 respectively downstream of Q499. The J339-z119 H chain gene of the anti-human F. X antibody was constructed by binding z119 downstream of J339.
[0269] [000269] Each of the antibody genes (Q499-z118, Q499-z121, J339-z119, and L377-k) was inserted into an animal cell expression vector.
[0270] [000270] The following antibodies were expressed transiently in FreeStyle293 cells (Invitrogen) by transfection using the constructed expression vectors. As shown below, the antibodies were named using the transfected antibody gene combinations. Q499-z118 / J339-z119 / L377-k Q499-z121 / J339-z119 / L377-k
[0271] [000271] The two antibodies above are only different at the amino acid of position 435 in the EU numbering system on the H chain of the anti-human F.IX antibody. z118 has His at position 435 and has protein A binding affinity. However, z121 has Arg at position 435, and is predicted to have no protein A binding activity based on the finding described in Example 2. Q499 is predicted to bind to protein A based on its sequence. Thus, as for Q499-z118 / J339-z119 / L377-k, the homomeric species J339-z119 / L377-k (a homomeric antibody against FX) has two protein A binding sites; the heteromeric antibody Q499-z118 / J339-z119 / L377-k has three; and the homomeric antibody Q499-z118 / L377-k (homomeric antibody against FIX) has four protein A binding sites. However, as for Q499-z121 / J339-z119 / L377-k introduced with a modification that leads to loss of protein A binding affinity, the J339-z119 / L377-k homomeric species has two protein A binding sites; the heteromeric antibody Q499-z121 / J339-z119 / L377-k has two; and the homomeric antibody Q499-z121 / L377-k has two. Specifically, even if a modification that leads to loss of protein A binding affinity (for example, a modification that replaces Arg with the amino acid at position 435, EU numbering) was introduced only in the H chain that binds protein A through its region variable, would not produce the effect that allows efficient isolation / purification of the heteromeric antibody at high purity by the purification step based on protein A alone. However, the modification that leads to loss of protein A binding capacity can produce the effect when MabSelct SuRe (GE Healthcare) is used. MabSelct SuRe is a modified protein A incapable of binding to Q499 and a chromatographic vehicle for use in the purification of antibodies. The vehicle was developed to satisfy conditions. The ligand is a recombinant protein A that has been modified by genetic engineering to be resistant to alkaline conditions. The great pH stability allows washing with efficient and low cost NaOH.
[0272] [000272] In addition, the vehicle is characteristic in that it does not connect to the variable region of the heavy chain of the VH3 subclass, such as Q499. With respect to Q499-z118 / J339-z119 / L377-k, the homomeric species J339-z119 / L377-k has two MabSelct SuRe binding sites; the heteromeric antibody Q499-z118 / J339-z119 / L377-k has two; and the homomeric antibody Q499-z118 / L377-k has two. However, as for Q499-z121 / J339-z119 / L377-k, the homomeric species J339-z119 / L377-k has two MabSelct SuRe binding sites; the heteromeric antibody Q499-z121 / J339-z119 / L377-k has a single site; and the homomeric antibody Q499-z121 / L377-k has no MabSelct SuRe binding site. Specifically, it is understood that by combining a modified protein A incapable of binding to the antibody variable region, such as MabSelct SuRe, with a modification that leads to loss of protein A binding affinity, the heteromeric antibody can be efficiently isolated and purified to high purity by the protein A-based purification step alone despite the protein A binding activity of the variable region of the heavy chain. [Example 6] Isolation and purification of heteromeric antibodies by affinity chromatography using modified protein A
[0273] [000273] The CM in which Q499-z118 / J339-z119 / L377-k or Q499-z121 / J339-z119 / L377-k had been expressed was subjected to chromatography using modified protein A. The CM samples were filtered through a 0.22 μm pore size filter, and loaded onto a Mab Select SuRe column (GE Healthcare) balanced with D-PBS. The column was subjected to washes 1 and 2 and eluted as shown in Table 7. Recombinant protein A consists of five domains (A to E) that have IgG binding activity. In Mab Select SuRe, domain B was modified by genetic engineering to have a tetrameric structure. Mab Select SuRe has no affinity for the antibody variable region, and is advantageous in that it allows antibody elution even under milder conditions compared to conventional recombinant protein A. In addition, the resin has improved alkaline resistance and allows cleaning in place using 0.1 to 0.5 M NaOH, and is therefore more suitable for production. In the experiment described in this Example as shown in Table 7, 50 mM acetic acid (the pH was not adjusted and the pH measured was approximately 3.0) was used for elution instead of gradual elution at pH 3.6 and pH 2.7 described in Example 3. The respective eluted fractions were collected and analyzed by cation exchange chromatography to identify their components. To prepare controls, each CM was loaded onto rProtein G Sepharose Fast Flow resin (GE Healthcare) in the same manner as described in Example 2. Samples purified by batch elution were used as controls.
[0274] [000274] Next, the eluted fractions of protein A were subjected to ion exchange chromatography. A Sepharose High Performance SP column (GE Healthcare) was equilibrated with an equilibration buffer (20 mM sodium phosphate buffer, pH 6.0). Then, eluted fractions of protein A were neutralized with 1.5 M Tris-HCl (pH7.4), and diluted three times with equilibration buffer, and loaded. Column-bound antibodies were eluted with 25 column volumes (CV) of a 50 to 350 mM NaCl concentration gradient. The eluted fractions containing heteromeric antibody were purified by gel filtration chromatography using superdex200. The resulting monomer fractions were collected, and used in the evaluation of pharmacokinetics in transgenic mice with human FcRn described in Example 7. Table 7
[0275] [000275] The result of the cation exchange chromatography analysis of each eluted fraction is shown in Tables 8 and 9. As shown in Table 8, with respect to Q499-z118 / J339-z119 / L377-k, the component ratio of each eluted fraction is not much different from that of the control. The reason is probably that the three species J339-z119 / L377-k (a homomeric antibody against FX), Q499-z118 / L377-k (a homomeric antibody against F.IX) and Q499-z118 / J339-z119 / L377 -k (a heteromeric antibody) have two modified protein binding sites, so there was no difference in association / dissociation during the protein A-based purification step.
[0276] [000276] However, in the case of Q499-z121 / J339-z119 / L377-k, the proportion of Q499-z121 / L377-k (a homomeric antibody against F.IX) in the eluted fraction was significantly reduced when compared to the control as shown in Table 9. In contrast, the proportions of J339-z119 / L377-k (a homomeric antibody against FX) and Q499-z121 / J339-z119 / L377-k (a heteromeric antibody) in the eluted fraction were relatively increased when compared control together with a reduction of Q499-z121 / L377-k. This is believed to be because J339-z119 / L377-k (a homomeric antibody against FX) has two modified protein A binding sites and Q499-z121 / J339-z119 / L377-k (a heteromeric antibody) has a. However, Q499-z121 / L377-k (a homomeric antibody against F.IX) has no binding site, and consequently the majority of Q499-z121 / L377-k passed through the column without binding to the modified protein A.
[0277] [000277] As described above, the present invention also demonstrates that with respect to antibodies whose variable regions have protein A binding activity, when the modified protein A is combined with a modification that leads to loss of protein A binding affinity , one of the homomeric antibodies can be significantly reduced, and as a result the purity of the heteromeric antibody is increased by the protein A-based purification step alone. Table 8
[0278] [000278] Q499-z118 / J339-z119 / L377-k and Q499-z121 / J339-z119 / L377-k prepared as described in Example 6 were evaluated for their pharmacokinetics.
[0279] [000279] It will probably be difficult to adjust protein A binding activity without loss of human FcRn binding, because protein A and human FcRn recognize the same site on an IgG antibody (J Immunol. 2000 164 (10): 5313- 8) as shown in Fig. 2. Preserving binding affinity for human FcRn is very important for long plasma retention (long half-life) in humans, which is characteristic of IgG-like antibodies. In this context, the pharmacokinetics were compared between Q499-z118 / J339-z119 / L377-k and Q499-z121 / J339-z119 / L377-k prepared as described in Example 6.
[0280] [000280] In a pharmacokinetic experiment to predict half-life in humans, the pharmacokinetics in transgenic mice with human FcRn (mice B6.mFcRn - / -. HFcRn strain Tg 276 + / +, Jackson Laboratories) was evaluated by the following procedure . Q499-z118 / J339-z119 / L377-k and Q499-z121 / J339-z119 / L377-k were each administered intravenously at a dose of 5 mg / kg to mice, and blood was collected at appropriate time. The collected blood was immediately centrifuged at 15,000 rpm and 4 ° C for 15 minutes to obtain blood plasma. The separated plasma was stored in a refrigerator at -20 ° C or below until use. The blood concentration was determined by ELISA.
[0281] [000281] As shown in Fig. 3, the result indicates that Q499-z118 / J339-z119 / L377-k and Q499-z121 / J339-z119 / L377-k were comparable to each other in terms of plasma retention. Thus, z121 / z119, a constant region into which any of the H chains is introduced with a modification that leads to loss of protein A binding capacity has been shown to be comparable in terms of plasma retention with z118 / z119 that does not have the modification which leads to loss of protein A binding affinity. As described above, the present inventors have disclosed a modification (for example, an Arg substitution mutation for amino acid at position 435, numbered EU) which leads to loss of the ability to bind to protein A but has no influence on pharmacokinetics, and it allows efficient isolation / purification of the heteromeric antibody at high purity by the purification step based on protein A alone despite the variable region. [Example 8] Introduction of mutations in the CH3 domain of GC33-IgG1-CD3-scFv and preparation of engineered molecules through the protein A-based purification step alone Introduction of purification mutations based on protein A of the GC33-IgG1-CD3-scFv molecule
[0282] [000282] The inventors designed an anti-GPC3 IgG antibody molecule in which an anti-CD3 scFv antibody is linked to one of the two H chains (Fig. 4). This molecule was expected to be able to kill cancer cells by recruiting T cells into cancer cells by divalent binding to glypican-3 (GPC3), a cancer-specific antigen, and monovalent binding to CD3, a T cell antigen. anti-CD3 scFv antibody must be linked to only one of the two H chains to achieve monovalent binding to CD3. In this case, it is necessary to purify the molecule formed through the heteromeric association of two types of H chains.
[0283] [000283] Thus, using the same method as described in Example 3, a substitution mutation from Arg to His at position 435 (EU numbering) was introduced into one of the H chains. In addition, the above mutation was combined with the mutations ( a substitution of Lys for Asp in position 356, numbered EU, is introduced in an H chain and a substitution of Glu for Lys in position 439, numbered EU, is introduced in another chain H) described in WO 2006/106905 (PROCESS FOR PRODUCTION OF POLYPEPTIDE BY REGULATION OF ASSEMBLY) as a modification to increase the heteromeric association of two H chains. The present inventors tested whether it was possible with the combined mutations to purify the molecule of interest by protein A chromatography alone. Construction of antibody gene expression vectors and expression of respective antibodies
[0284] [000284] The gene encoding GPC3 (anti-human Glipicano-3 antibody H chain variable region, SEQ ID NO: 22) as an antibody H chain variable region was constructed by a method known to those skilled in the art. In addition, the gene encoding GC33-k0 (anti-human Glipicano-3 antibody L chain, SEQ ID NO: 23) as an antibody L chain was constructed by a method known to those skilled in the art. In addition, the genes described below were constructed as an antibody H chain constant region by a method known to those skilled in the art. LALA-G1d (SEQ ID NO: 24), which was constructed from IgG1 replacing Ala with Leu at positions 234 and 235 (EU numbering), and Ala with Asn at position 297 (EU numbering), and elimination of Gly and Lys C- terminals LALA-G1d-CD3 (SEQ ID NO: 25), which was constructed from LALA-G1D linking an anti-CD3 scFv (in which the H chain variable region of the anti-human CD3 antibody is linked via a peptide linker to the terminal C of the L chain variable region of the anti-human CD3 antibody) LALA-G3S3E-G1d (SEQ ID NO: 26), which was constructed from LALA-G1D replacing Arg with His in position 435 (EU numbering) and Glu with Lys in position 439 (EU numbering); and LALA-S3K-G 1d-CD3 (SEQ ID NO: 27), which was constructed from LALA-G1D-CD3 replacing Lys with Asp at position 356 (EU numbering).
[0285] [000285] The H chain NTA1L and NTA1R genes of the anti-human GPC3 antibody were constructed by linking respectively LALA-G1d-CD3 (in which an anti-CD3 scFv antibody is linked to the H chain constant region) and LALA-G1d (a region chain constant) downstream of GPC3, which is the variable region of the H chain of an anti-human Glipicano-3 antibody. In addition, the anti-human GPC3 antibody H chain NTA2L and NTA2R genes were constructed by linking an anti-CD3 scFv antibody downstream of GPC3 as a H chain constant region, and linking LALA-S3K-G1d-CD3 introduced with a mutation of substitution of Lys for Asp in position 356 (numbering EU) or LALA-G3S3E-G1d introduced with mutations of substitution of Arg for His in position 435 (numbering EU) and Glu for Lys in position 439 (numbering EU). The constructed genes were listed below. H chain NTA1L ・ F GPC3-LALA-G1d-CD3 NTA1R ・ F GPC3-LALA-G1d NTA2L ・ F GPC3-LALA-S3K-G1d-CD3 NTA2R ・ F GPC3-LALA-G3S3E-G1d L chain GC33-k0
[0286] [000286] Each of the antibody genes (H chains: NTA1L, NTA1R, NTA2L, and NTA2R; L chain: GC33-k0) was inserted into an animal cell expression vector. Using a method known to those skilled in the art, the antibodies listed below were transiently expressed in FreeStyle293 cells (Invitrogen) by transfecting the cells with the constructed expression vectors. As shown below, the antibodies were named using the transfected antibody gene combinations (first H chain / second H chain / L chain). NTA1L / NTA1R / GC33-k0 NTA2L / NTA2R / GC33-k0 Protein purification of the expressed samples and evaluation of heterodimer yield
[0287] [000287] FreeStyle293 (CM) cell culture supernatants containing the following antibodies were used as a sample. NTA1L / NTA1R / GC33-k0 NTA2L / NTA2R / GC33-k0
[0288] [000288] The CM samples were filtered through a filter with a pore size of 0.22 μm, and loaded onto a rProtein A Sepharose Fast Flow column (GE Healthcare) equilibrated with D-PBS. The column was subjected to washes 1 and 2 and elution 1 as shown in Table 10. The volume of the CM to be loaded onto the column was adjusted to 20 mg antibody / ml of resin. The respective fractions eluted under each condition were collected and analyzed by size exclusion chromatography to identify its components. Table 10
[0289] [000289] The result of the size exclusion chromatography of each eluted fraction is shown in Fig. 5 and Table 11 below. The values represent the peak elution area expressed as a percentage. For NTA1L / NTA1 R / GC33-k0 and NTA2L / NTA2R / GC33-k0, homomeric antibodies (antibodies with homomeric NTA1L or homomeric NTA2L) that have the anti-CD3 scFv antibody in both chains were barely detectable. This is thought to be caused by the extremely low level of expression of the H chains containing the scFv anti-CD3 antibody because the level of expression of a scFv molecule is generally low. As for homomeric antibodies that do not contain the scFv anti-CD3 antibody in its two chains, approximately 76% of the homomeric antibody NTA1R was observed in the case of NTA1L / NTA1R / GC33-k0, while only approximately 2% of the homomeric NTA2R antibody was observed in the case of NTA2L / NTA2R / GC33-k0. Thus, the present invention demonstrated that when the substitution mutations of Lys for Glu at position 356 (EU numbering) and Glu for Lys at position 439 (EU numbering) for the efficient formation of heteromeric molecules of the respective H chains, were combined with the Arg to His substitution mutation at position 435 (EU numbering), the heteromeric antibody (bispecific antibody of interest) can be efficiently purified to a purity of 98% or more by the protein A-based purification step alone. Table 11
[0290] [000290] An ordinary anti-GPC3 IgG antibody divally binds via two H chains to glypican-3 (GPC3), a cancer-specific antigen. In the experiment described in this Example, the inventors designed and evaluated an anti-GPC3 IgG antibody molecule (Fig. 6) that monovalently binds to glypican-3. It is believed that when compared to ordinary divalent antibodies, the molecule's monovalent binding to glypican-3 (GPC3), a cancer-specific antigen, was based on affinity rather than greed. Thus, it was expected that the molecule would be able to bind to the antigen without crosslinking. To achieve the monovalent bonding of two H chains to glypican-3 (GPC3), each must be an H chain consisting of a hinge-Fc domain that lacks the variable region and CH1 domain, while the other is an H chain ordinary. In this case, it is necessary to purify the molecule that results from the heteromeric association of two types of H chains.
[0291] [000291] Thus, using the same method as described in Example 3, a substitution mutation from Arg to His at position 435 (EU numbering) was introduced into one of the H chains. In addition, the above mutation was combined with the mutations (a substitution of Lys for Asp in position 356, numbered EU, is introduced in an H chain and a substitution of Glu for Lys in position 439, numbered EU, is introduced in another chain H) described in WO 2006/106905 (PROCESS FOR PRODUCTION OF POLYPEPTIDE BY REGULATION OF ASSEMBLY) as a modification to increase the heteromeric association of two H chains. The present inventors evaluated whether it was possible with the combined mutations to purify the molecule of interest by protein A chromatography alone. Construction of antibody gene expression vectors and expression of the respective antibodies
[0292] [000292] The variable region of the H chain of the antibody used was: GPC3 (the H chain variable region of an anti-human Glipican-3 antibody, SEQ ID NO: 22).
[0293] [000293] The antibody L chain used was: GC33-k0 (the L chain of an anti-human Glipican-3 antibody, SEQ ID NO: 23).
[0294] [000294] The antibody H chain constant regions used were: LALA-G1d (SEQ ID NO: 24), which was constructed of IgG1 introducing Leu substitution mutations at positions 234 and 235 (EU numbering), and Ala by Asn at position 297 (EU numbering), and elimination of Gly and Lys C-terminals; LALA-G3-G1d (SEQ ID NO: 28), which was constructed from LALA-G1D by introducing an Arg to His mutation at position 435 (EU numbering); LALA-G3S3E-G1d (SEQ ID NO: 26), which was constructed from LALA-G3-G1D introducing a Lys substitution mutation from Glu at position 439 (EU numbering); LALA-G1Fc (SEQ ID NO: 2), which was constructed from LALA-G1D deleting positions 1 to 215 (EU numbering); and LALA-G1Fc-S3K (SEQ ID NO: 30), which was constructed from G1Fc introducing an Asp substitution mutation of Lys at position 356 (EU numbering).
[0295] [000295] The NTA4L-cont, NTL4L-G3, and NTA4L genes of the anti-human GPC3 antibody H chain were constructed by binding downstream of GPC3 (the variable chain H variable of an anti-human Glipican-3 antibody) , respectively, LALA-G1d (a constant H region chain), LALA-G3-G1d introduced with an Arg substitution mutation at His at position 435 (EU numbering), and LALA-G3S3E-G1d introduced with Arg substitution mutations by His at position 435 (EU numbering) and Glu by Lys at position 439 (EU numbering). In addition, the FTA genes NTA4R-cont and NTA4R were constructed using LALA-G1Fc (an anti-human hinge Fc domain) and LALA-G1Fc-S3K (a hinge Fc domain introduced with an Asp Lys replacement mutation in position 356, EU numbering). The constructed genes are: H chain NTA4L-cont ・ F GPC3-LALA-G1d NTA4L-G3 ・ F GPC3-LALA-G3-G1d NTA4L ・ F GPC3-LALA-G3S3E-G1 d NTA4R-cont: LALA-G1Fc NTA4R: LALA-G1Fc-S3K L chain GC33-k0
[0296] [000296] The antibody genes (NTA4L, NTA4L-cont, NTA4L-G3, NTA4R, NTA4R-cont, and GC33-k0) were each inserted into an animal cell expression vector.
[0297] [000297] The following antibodies were transiently expressed in FreeStyle293 cells (Invitrogen) by transfection using the constructed expression vectors. As shown below, the antibodies were named using the transfected antibody gene combinations. NTA4L-cont / NTA4R-cont / GC33-k0 NTA4L-G3 / NTA4R-cont / GC33-k0 NTA4L / NTA4R / GC33-k0 Protein purification of expressed samples and evaluation of heterodimer yield
[0298] [000298] The CM containing the following antibody was used as a sample: NTA4L-cont / NTA4R-cont / GC33-k0 NTA4L-G3 / NTA4R-cont / GC33-k0 NTA4L / NTA4R / GC33-k0
[0299] [000299] The CM samples were filtered through a filter with a pore size of 0.22 μm, and loaded onto a rProtein A Sepharose Fast Flow column (GE Healthcare) equilibrated with D-PBS. The column was subjected to washes 1 and 2 and elution 1 as shown in Table 12. The volume of the CM to be loaded onto the column was adjusted to 20 mg antibody / ml of resin. The respective fractions eluted under each condition were collected and analyzed by size exclusion chromatography to identify its components. Table 12
[0300] [000300] The result of the chromatography analysis by size exclusion of each eluted fraction is shown in Fig. 7 and Table 13 below. The values represent the peak elution area expressed as a percentage.
[0301] [000301] As for NTA4L-cont / NTA4R-cont / GC33-k0, the homomeric antibody that divally binds to GPC3 (homomeric antibody NTA4L-cont) and the homomeric molecule that has no GPC3-binding domain (homomeric antibody NTA4R- cont) was eluted, while the heteromeric antibody of interest, NTA4L-cont / NTA4R-cont, accounted for only 46.5%.
[0302] [000302] In the case of NTA4L-G3 / NTA4R-cont / GC33-k0, the homomeric antibody that divally binds to GPC3 (homomeric antibody NTA4L-G3) was barely detectable, while the homomeric molecule that has no binding domain to GPC3 (homomeric antibody NTA4R-cont) was abundant. The heteromeric antibody of interest, NTA4L-G3 / NTA4R-cont, accounted for 66.7%. In the case of NTA4L / NTA4R / GC33-k0, the homomeric antibody that divally binds to GPC3 (homomeric antibody NTA4L) was barely detectable, and the proportion of the homomeric molecule that has no GPC3-binding domain (NTA4R) was considerably reduced , resulting in a significant increase of up to 93.0% in the proportion of the heteromeric antibody of interest, NTA4L / NTA4R. Thus, the present invention demonstrated that when the substitution mutations of Lys by Asp at position 356 (EU numbering) and Glu by Lys at position 439 (EU numbering) for the efficient formation of heteromeric molecules of the respective H chains were introduced in In combination with the Arg to His substitution mutation at position 435 (EU numbering), the heteromeric antibody (a bispecific antibody of interest) can be efficiently purified to a purity of 93% or more by the protein A-based purification step alone. Table 13
[0303] [000303] As described in Example 9, the present inventors demonstrated that in the case of an antibody having the variable region in only one arm, the heteromeric antibody can be efficiently purified by the protein A-based purification step alone by combining the substitution mutation of Arg by His in position 435 (EU numbering) with the mutations (a replacement of Lys by Asp in position 356, EU numbering, is introduced in an H chain and a replacement of Glu by Lys in position 439, EU numbering, is introduced in another H chain) described in WO 2006/106905 (PROCESS FOR PRODUCTION OF POLYPEPTIDE BY REGULATION OF ASSEMBLY). However, the heteromeric antibody is not purified to a sufficiently high purity with elution 1 (elution buffer: 2 mM HCl, pH 2.7) alone. An additional purification step is required.
[0304] [000304] Then, in this Example, the present inventors have evaluated whether the heteromeric antibody can be isolated and purified to high purity by protein A column chromatography using elution with a pH gradient. This was based on the assumption that more protein A binding sites lead to stronger binding of the heteromeric antibody to protein A, and as a result, lower pH is required for elution. Purification can be achieved more efficiently at a lower cost when the purity of the heteromeric antibody can be increased to almost 100% using such a pH gradient elution.
[0305] [000305] CM samples containing the following antibodies were used: NTA4L-cont / NTA4R-cont / GC33-k0 NTA4L-G3 / NTA4R-cont / GC33-k0 NTA4L / NTA4R / GC33-k0
[0306] [000306] CM samples were filtered through a filter with a pore size of 0.22 μm, and loaded onto a HP HiTrap protein A column (GE Healthcare) balanced with D-PBS. The column was subjected to washes 1 and 2 successively, and then eluted with a pH gradient using elution A and B as shown in Table 14. Elution of pH gradient was achieved with the following linear gradient: elution A / elution B = (100: 0) ↑ (30:70) for 35 minutes. The eluted fractions were collected and analyzed by size exclusion chromatography analysis to identify their components. Table 14
[0307] [000307] NTA4L-cont / NTA4R-cont / GC33-k0, NTA4L-G3 / NTA4R-cont / GC33-k0 and NTA4L / NTA4R / GC33-k0 were purified by protein A column chromatography under condition of gradient elution pH. The resulting chromatograms are shown in Fig. 8. Elution of NTA4L-cont / NTA4R-cont / GC33-k0 resulted in a large peak. However, NTA4L-G3 / NTA4R-cont / GC33-k0 pH gradient elution provided two elution peaks. The high and low pH peaks were marked as "elution 1" and "elution 2", respectively. The result for NTA4L / NTA4R / GC33-k0 was approximately the same as that for NTA4L-G3 / NTA4R-cont / GC33-k0, except that the peak elution area 2 was smaller.
[0308] [000308] The result of the size exclusion chromatography analysis for each peak is shown in Table 15. NTA4L-cont / NTA4R-cont / GC33-k0 provided three components eluted in this order: a homomeric antibody that divally binds to GPC3 ( homomeric antibody NTA4L-cont), a heteromeric antibody that monovalently binds to GPC3 (heteromeric antibody NTA4L-cont / NTA4R-conc), and a homomeric molecule that has no GPC3 binding domain (homomeric antibody NTA4R-cont). It is believed that the reason why these components were not separated by pH gradient elution is that they have the same number (two) of protein A binding sites. However, it was revealed that in elution 1 of NTA4L-G3 / NTA4R -cont / GC33-k0, the levels of the homomeric antibody that divally binds to GPC3 (homomeric antibody NTA4L-G3) and the homomeric molecule that has no binding domain to GPC3 (homomeric antibody NTA4R-cont) were below the detection limit, while the heteromeric antibody that monovalently binds to GPC3 (antibody NTA4L-G3 / NTA4R-conc heteromeric) accounted for 99.6%. In elution 2, it was found that the homomeric molecule that has no GPC3 binding domain (homomeric antibody NTA4R-cont) accounted for 98.8%. The homomeric NTA4L-G3 antibody passes through the protein A column because it cannot bind to protein A due to the Arg to His substitution mutation at position 435 (EU numbering). However, the heteromeric antibody NTA4L-G3 / NTA4R-conc has a unique protein A binding site, while the homomeric antibody NTA4R-cont has two. More protein A binding sites mean stronger protein A binding, and as a result, lower pH was required for elution. This is thought to be the reason why the homomeric antibody NTA4R-cont was eluted at a lower pH than the heteromeric antibody NTA4L-G3 / NTA4R-conc. Almost the same result was obtained for NTA4L / NTA4R / GC33-k0. The result of the size exclusion chromatography analysis shows that the proportion of components was comparable to that of NTA4L-G3 / NTA4R-cont / GC33-k0. There was a difference between protein A chromatograms, and the ratio of peak area from elution 2 to elution 1 was lower in NTA4L / NTA4R / GC33-k0. The proportion of expression of the homomeric antibody NTA4R-cont, which is the main component of elution 2, was reduced due to the mutations introduced for efficient generation of the heteromeric antibody NTA4L-G3 / NTA4R-conc. The amino acid mutations described above improved the purification yield of the heteromeric antibody and the robustness of purification by protein chromatography column chromatography with pH gradient elution.
[0309] [000309] As described above, the present inventors have demonstrated that the heteromeric antibody can be efficiently isolated and purified to high purity by the purification step using protein A column chromatography alone with pH gradient elution. Table 15
[0310] [000310] Introduction of mutation in the CH3 domain and preparation of monovalent Fcalfa receptor-Fc fusion protein by the purification step based on protein A
[0311] [000311] Conventional Fc-Fc receptor fusion proteins, such as Eternercept and Abatacept are homodimers that can divally bind to ligands. In the experiment described in this Example, the inventors designed and evaluated an Fc-Fc receptor fusion protein that monovalently binds to IgA as a ligand (Fig. 9). To achieve monovalent binding of the Fcalfa receptor to IgA, one of the two H chains of the Fc-Fc receptor fusion protein must be the entire H chain having the Fc-hinge domain. In this case, it is necessary to purify the molecule that results from the heteromeric association of two types of H chains
[0312] [000312] Thus, using the same method as described in Example 6, a substitution mutation from Arg to His at position 435 (EU numbering) was introduced into one of the two H chains. In addition, the above mutation was combined with mutations ( a substitution of Lys for Asp in position 356, numbering EU, is introduced in an H chain and a substitution of Glu for Lys in position 439, EU numbering is introduced in another chain H) described in WO 2006/106905 (PROCESS FOR PRODUCTION OF POLYPEPTIDE BY REGULATION OF ASSEMBLY) as a modification to increase the heteromeric association of two types of H chains. The present inventors evaluated whether it was possible with the combined mutations to purify the molecule of interest by protein A chromatography alone. Construction of antibody gene expression vectors and expression of respective antibodies
[0313] [000313] The Fc receptor used was FcalfaR (human IgA1 receptor, SEQ ID NO: 31). The constant regions of the fusion H chain used were: G1Fc (SEQ ID NO: 32), which is a human hinge-Fc domain constructed of IgG1 deleting Gly and Lys C-terminals, and residues from positions 1 to 223 (EU numbering); G1Fc-G3S3K (SEQ ID NO: 33), which was constructed from G1Fc introducing mutations for replacing Lys by Asp at position 356 (EU numbering) and Arg by His at position 435 (EU numbering); and G1Fc-S3E (SEQ ID NO: 34), which was constructed from G1Fc introducing a Glu substitution mutation by Lys at position 439 (EU numbering).
[0314] [000314] The FcalfaR-Fc fusion proteins IAL-cont and IAL were constructed by ligating downstream of FcalfaR through a polypeptide linker (SEQ ID NO: 35), G1Fc (a constant region of the H chain) and G1Fc-G3S3K introduced with substitution mutations of Lys for Asp at position 356 (EU numbering) and Arg for His at position 435 (EU numbering).
[0315] [000315] In addition, the Fc genes IAR-cont and IAR were constructed to encode G1Fc (a human Fc hinge domain) and G1Fc-S3E (an Fc hinge domain introduced with a Lys-substituted Glu mutation at position 439 , EU numbering), respectively. The constructed genes were: H chain IAL-cont ・ F FcalfaR-G1Fc IAL ・ F FcalfaR-G1Fc-G3S3K IAR-cont: G1Fc IAR: G1Fc-S3E
[0316] [000316] The antibody genes (IAL-cont, IAL, IAR-cont, and IAR) were each inserted into an animal cell expression vector.
[0317] [000317] The following antibodies were expressed transiently in FreeStyle293 cells (Invitrogen) by transfection using the constructed expression vectors. As shown below, the antibodies were named using the transfected antibody gene combinations. IAL-cont / IAR-cont IAL / IAR Purification of express sample proteins and evaluation of heterodimer yield CM samples containing the following antibody were used: IAL-cont / IAR-cont IAL / IAR
[0318] [000318] The CM samples were filtered through a filter with a pore size of 0.22 μm, and loaded onto a rProtein A Sepharose Fast Flow column (GE Healthcare) equilibrated with D-PBS. The column was subjected to washes 1 and 2 and elution 1 as shown in Table 16. The volume of the CM to be loaded onto the column was adjusted to 20 mg antibody / ml of resin. The respective fractions eluted under each condition were collected and analyzed by size exclusion chromatography to identify its components. Table 16
[0319] [000319] The result of the size exclusion chromatography analysis of each eluted fraction is shown in Fig. 10 and Table 17 below. The values represent the peak elution area expressed as a percentage. As for IAL-cont / IAR-cont, a homomeric antibody that divally binds to IgA (homomeric antibody IAL-cont) and a homomeric molecule that has no IgA binding site (homomeric antibody IAR-cont) was eluted, while the heteromeric antibody IAL-cont / IAR-cont of interest accounted for only 30%. In the case of IAL / IAR, the homomeric antibody that divally binds to IgA (homomeric antibody IAL) was not detectable, and the proportion of the homomeric molecule that has no IgA-binding site (homomeric antibody IAR) was considerably reduced; thus, the heteromeric antibody IAL / IAR of interest was significantly increased to approximately 96%. Thus, the present invention demonstrated that when the substitution mutations of Lys by Asp at position 356 (EU numbering) and Glu by Lys at position 439 (EU numbering) for the efficient formation of heteromeric molecules of the respective H chains were introduced in In combination with the Arg to His substitution mutation at position 435 (EU numbering), the heteromeric antibody, bispecific antibody of interest, can be efficiently purified to a purity of 95% or more by the protein A-based purification step alone. Table 17
[0320] [000320] The bispecific antibody against human F.IX and human F.X, which was designed as described in Example 1, consists of a common L chain and two types of H chains in which each recognizes a different antigen. Obtaining a bispecific antibody with a common L chain is not easy, because it is difficult for a common L chain sequence to recognize two different types of antigens. As described above, obtaining a common L chain is extremely difficult. Thus, it can be suspected that a more preferred option is a bispecific antibody consisting of two types of H chains and two types of L chains that recognize two types of antigens. If two types of H chains and two types of L chains are expressed, they will form ten types of IgG H2L2 molecules in random combinations. It is very difficult to purify the bispecific antibody of interest from ten types of antibodies.
[0321] [000321] In the experiment described in this Example, the present inventors prepared and evaluated bispecific antibodies consisting of two types of H chains and two types of L chains against human IL-6 receptor and human glypican-3 (GPC3). To efficiently prepare bispecific antibodies consisting of two types of H chains and two types of L chains, it is necessary to increase the association of H chains and L chains against the same antigen as well as the heteromeric association of two types of H chains. It is essential that the bispecific antibody with the right combination can be purified from the obtained expression products.
[0322] [000322] To increase the association between H chains and L chains against the same antigen, the variable region (VH) of the H chain (GC33-VH-CH1-hinge-CH2-CH3) and the variable region (VL) of the L chain (GC33-VL-CL) from GC33 (an anti-GPC3 antibody) were exchanged with each other to produce the H chain GC33-VL-CH1-hinge-CH2-CH3 and L chain (GC33-VH-CL) (the VH domain and VL domain were exchanged). GC33-VL-CH1-hinge-CH2-CH3 associates with GC33-VH-CL; however, its association with the L chain (MRA-VL-CL) of the anti-IL-6 receptor antibody is inhibited due to the instability of the VL / VL interaction. Likewise, the H chain (MRA-VH-CH1-hinge-CH2-CH3) of the anti-IL-6 receptor antibody associates with MRA-VL-CL; however, its association with the L chain (GC33-VH-CL) of the anti-GPC3 antibody is inhibited due to the instability of the VH / VH interaction. As described above, it is possible to increase the association between H chains and L chains against the same antigen. However, the VH / VH interaction and the VL / VL interaction also occur although they are less stable than the FEH Lett VH / VL interaction. 2003 Nov 20, 554 (3): 323-9; J Mol Biol. 2003 Oct 17, 333 (2): 355-65; for VL / VL, see: J Struct Biol. 2002 Jun, 138 (3): 171-86; Proc Natl Acad Sci USA. 1985 Jul, 82 (14): 4592-6), and thus although not often, even the association of H chains and L chains also occurs unfavorably. Therefore, although the percentage of the bispecific antibody of interest is increased by simply exchanging the VH domain and the VL domain with each other, the expressed products still contain approximately ten types of combinations.
[0323] [000323] In general, it is extremely difficult to purify the bispecific antibody of interest of ten types. However, it is possible to improve the separation of ten types of components in ion exchange chromatography by introducing a modification so that ten types of components each have a different isoelectric point. In this context, MRA-VH, which is the H chain variable region of an anti-IL-6 receptor antibody, was modified to lower the isoelectric point, and this produced H54-VH with a lower isoelectric point. In the same way, MRA-VL, which is the L-chain variable region of an anti-IL-6 receptor antibody, was modified to lower the isoelectric point, and this produced L28-VL with a lower isoelectric point. In addition, GC33-VH, which is the H chain variable region of an anti-GPC3 antibody, has been modified to increase the isoelectric point. This produced Hu22-VH with an increased isoelectric point.
[0324] [000324] The combination of the H and L chains of interest was improved by exchanging the VH and VL between the H chains and the L chains of the anti-GPC3 antibody. However, although not frequently, the unfavorable H chain / L chain association occurs because it is impossible to completely suppress the H54-VH / Hu22-VH interaction and the L28-VL / GC33-VL interaction. An ordinary antibody sequence has glutamine at position 39 in VH. In the VH / VH interaction, it is believed that glutamines form hydrogen bonds at the VH / VH interface. Then, lysine was replaced by glutamine at position 39 (Kabat numbering) to impair H54-VH / Hu22-VH interaction. It is thus expected that the VH / VH interaction would be significantly impaired due to the electrostatic repulsion between two lysines at the VH / VH interface. Next, H54-VH-Q39K and Hu22-VH-Q39K were constructed by replacing lysine with glutamine at position 39 (Kabat numbering) in the sequences of H54-VH and Hu22-VH. Likewise, an ordinary antibody sequence has glutamine at position 38 in VL. In the VL / VL interaction, glutamines are expected to form hydrogen bonds at the VL / VL interface. Then, glutamic acid was replaced by glutamine at position 38 (Kabat numbering) to impair the L28-VL / GC33-VL interaction. It is thus expected that the VL / VL interaction would be significantly impaired due to the electrostatic repulsion between two glutamic acids at the VL / VL interface. Afterwards, L28-VL-Q38E and GC33-VL-Q38E were constructed by substituting glutamic acid for glutamine in position 39 (Kabat numbering) in the sequences of L28-VL and GC33-VL.
[0325] [000325] To further improve the efficiency of expression / purification of the bispecific antibody of interest, an Arg to His substitution mutation at position 435 (EU numbering) was introduced into an H chain using the same method described in Example 3. In addition , the above mutation has been combined with the mutations (a substitution of Lys for Asp at position 356, EU numbering, is introduced in an H chain and a substitution of Glu for Lys at position 439, EU numbering, is introduced in another H chain) described in WO 2006/106905 (PROCESS FOR PRODUCTION OF POLYPEPTIDE BY REGULATION OF ASSEMBLY) as a modification to increase the heteromeric association of two types of H chains. The combined mutations allow the purification of the molecule that results from the heteromeric association of two types of chains. H by protein A chromatography alone.
[0326] [000326] Specifically, the H chain variable regions of the antibody used were: MRA-VH (the H chain variable region of an anti-human interleukin-6 receptor antibody, SEQ ID NO: 36); GC33-VH (the H chain variable region of an anti-GPC3 antibody, SEQ ID NO: 37); H54-VH (the H chain variable region of an anti-human interleukin-6 receptor antibody, SEQ ID NO: 38) with an isoelectric point lower than that of MRA-VH; Hu22-VH (the H chain variable region of an anti-GPC3 antibody, SEQ ID NO: 39) with an isoelectric point higher than that of GC33-VH; H54-VH-Q39K (SEQ ID NO: 40) where Lys is replaced by Gln at position 39 (Kabat numbering) following H54-VH; and Hu22-VH-Q39K (SEQ ID NO: 41) where Lys is replaced by Gln at position 39 in the sequence of Hu22-VH.
[0327] [000327] The following antibody H chain constant regions were also used: IgG1-LALA-N297A-CH (SEQ ID NO: 42) where Ala is replaced by Leu at positions 234 and 235 (EU numbering), and Ala is replaced by Asn at position 297 (EU numbering), and Gly and Lys C- terminals are deleted in the sequence of the IgG1 H chain constant region; IgG1-LALA-N297A-CHr (SEQ ID NO: 43) where the IgG1-LALA-N297A-CH sequence has two extra residues of Ser at the N-terminus; IgG1-LALA-N297A-s3-CH (SEQ ID NO: 44) where Glu is replaced by Lys at position 439 (EU numbering) following IgG1-LALA-N297A-CH; and IgG1-LALA-N297A-G3s3-CHr (SEQ ID NO: 45) where Lys is replaced by Asp at position 356 (EU numbering) and Arg is replaced by His at position 435 (EU numbering) following IgG1-LALA-N297A -CHr.
[0328] [000328] However, the antibody L variable chain regions used were: MRA-VL (the L chain variable region of an anti-human interleukin-6 receptor antibody, SEQ ID NO: 46); GC33-VL (the L-chain variable region of an anti-GPC3 antibody, SEQ ID NO: 47); L28-VL (the L-chain variable region of an anti-human interleukin-6 antibody, SEQ ID NO: 48) with an isoelectric point lower than that of MRA-VL; L28-VL-Q38E (SEQ ID NO: 49) where Glu is replaced by Gln at position 38 (Kabat numbering) in the sequence of L28-VL; and GC33-VL-Q38E (SEQ ID NO: 50) where Glu is replaced by Gln at position 38 (Kabat numbering) in the sequence of GC33-VL.
[0329] [000329] The following antibody L chain constant regions were also used. IgG1-CL (the IgG1 L chain constant region, SEQ ID NO: 51). IgG1-CLr (SEQ ID NO: 52), which was constructed by replacing Arg and Thr with Ala and Ser C-terminals, respectively, in the sequence of IgG1-CL. No1-Mh-H gene was constructed by ligating IgG1-LALA-N297A-CH downstream of MRA-VH. No1-Mh-L gene was constructed by ligating IgG1-CL downstream of MRA-VL. Gene no1-Gh-H was constructed by ligating IgG1-LALA-N297A-CH downstream of GC33-VH. Gene no1-Gh-L was constructed by ligating IgG1-CL downstream of GC33-VL. Gene no2-Gh-H was constructed by ligating IgG1-LALA-N297A-CHr downstream of GC33-VL. Gene no2-Gh-L was constructed by ligating IgG1-CLr downstream of GC33-VH. Gene no3-Ml-H was constructed by ligating IgG1-LALA-N297A-CH downstream of H54-VH. Gene no3-Ml-L was constructed by ligating IgG1-CL downstream of L28-VL. Gene no3-Ghh-L was constructed by ligating IgG1-CLr downstream of Hu22-VH. Gene no5-Ml-H was constructed by ligating IgG1-LALA-N297A-s3-CH downstream of H54-VH. Gene no5-Gh-H was constructed by ligating IgGl -LALA-N297A-G3s3-CHr downstream of GC33-VL. Gene # 6-Ml-H was constructed by ligating IgG1-LALA-N297A-s3-CH downstream of H54-VH-Q39K. Gene no6-Ml-L was constructed by ligating IgG1-CL downstream of L28-VL-Q38E. Gene no6-Gh-H was constructed by ligating IgG1-LALA-N297A-G3s3-CHr downstream of GC33-VL-Q38E. Gene no6-Ghh-L was constructed by ligating IgG1-CLr downstream of Hu22-VH-Q39K.
[0330] [000330] The respective genes (no1-Mh-H, no1-Mh-L, no1-Gh-H, no1-Gh-L, no2-Gh-H, no2-Gh-L, no3-Ml-H, no3 -Ml-L, no3-Ghh-L, no5-Ml-H, no5-Gh-H, no6-Ml-H, no6-Ml-L, no6-Gh-H, and no6-Ghh-L) were inserted in animal cell expression vectors.
[0331] [000331] The following combinations of expression vectors were introduced into FreeStyle293-F cells to transiently express each projected molecule. A. Projected molecule: no1 (Fig. 11)
[0332] [000332] Description: anti-natural IL-6 receptor antibody / bispecific anti-GPC3. The polypeptides encoded by polynucleotides inserted in the expression vector: no1-Mh-H (SEQ ID NO: 53), no1-Mh-L (SEQ ID NO: 54), no1-Gh-H (SEQ ID NO: 55), and no1-Gh-L (SEQ ID NO: 56). B. Projected molecule: no2 (Fig. 12)
[0333] [000333] Description: constructed from no1 exchanging the VH and VL domains of the anti-GPC3 antibody.
[0334] [000334] Polypeptides encoded by polynucleotides inserted into the expression vector: no1-Mh-H, no1-Mh-L, no2-Gh-H (SEQ ID NO: 57), and no2-Gh-L (SEQ ID NO: 58). C. Projected molecule: no3 (Fig. 13)
[0335] [000335] Description: constructed of no2 introducing modifications to each chain to change its isoelectric point.
[0336] [000336] Polypeptides encoded by polynucleotides inserted into the expression vector: no3-Ml-H (SEQ ID NO: 59), no3-Ml-L (SEQ ID NO: 60), and no2-Gh-H, and no3- Ghh-L (SEQ ID NO: 61). D. Projected molecule: no5 (Fig. 14)
[0337] [000337] Description: constructed from no3 introducing a modification to increase heteromeric H chain association and a modification that allows protein A based purification of the antibody generated via heteromeric association.
[0338] [000338] Polypeptides encoded by polynucleotides inserted into the expression vector: no5-Ml-H (SEQ ID NO: 62), no3-Ml-L, no5-Gh-H (SEQ ID NO: 63), and no3-Ghh -L. E. Projected molecule: no6 (Fig. 15)
[0339] [000339] Description: constructed from no5 introducing a modification to increase the association between an H chain of interest and an L chain of interest.
[0340] [000340] Polypeptides encoded by polynucleotides inserted into the expression vector: no6-Ml-H (SEQ ID NO: 64), no6-Ml-L (SEQ ID NO: 65), no6-Gh-H (SEQ ID NO: 66), and no6-Ghh-L (SEQ ID NO: 67).
[0341] [000341] Culture supernatants filtered through a filter with a pore size of 0.22 pm were loaded onto the rProtein A Sepharose Fast Flow resin (GE Healthcare) balanced with the medium. The resin was eluted in a batch manner to purify the molecules. Since protein G binds to the Fab domain of an antibody, all antibody species in CM can be purified with protein G despite affinity for protein A.
[0342] [000342] The projected antibodies (no1, no2, no3, no5, and no6) were evaluated for their expression by cation exchange chromatography (IEC) using a WCX-10 ProPac column (Dionex), an analytical column. The cation exchange chromatography was performed at a flow rate of 0.5 ml / min with a suitable gradient using mobile phase A (MES-NaOH 20 mM, pH 6.1) and mobile phase B (MES-NaOH 20 mM, 250 mM NaCl, pH 6.1). The result of the IEC evaluation of each antibody is shown in Fig. 16. Natural anti-IL-6 receptor / bispecific anti-GPC3 antibody no1 provided several peaks in close proximity to each other. It was impossible to determine which peak corresponds to the bispecific antibody of interest. The same applied to no2 resulting from the exchange of the VH domain and VL domain of the anti-GPC3 antibody in no1. The peak of the bispecific antibody of interest can be isolated for the first time at no3 which has been modified from no2 by introducing a modification to alter the isoelectric point of each no2 chain. The proportion of the peak corresponding to the bispecific antibody of interest was significantly increased by no5 which was constructed from no3 introducing a modification to increase the heteromeric association of the H chain and a modification that allows protein A based purification of the antibody generated via heteromeric association. The proportion of the peak corresponding to the bispecific antibody of interest was further increased by no6 which was constructed from no5 introducing a modification that increases the association between the H chain and the L chain of interest.
[0343] [000343] Then, the present inventors have evaluated whether the bispecific antibody of interest can be purified from CM # 6 to high purity using a purification column. The CM samples were filtered through a filter with a pore size of 0.22 μm and loaded onto a HiTrap protein A HP column (GE Healthcare) equilibrated with D-PBS. The column was successively subjected to washes 1 and 2 and eluted with a pH gradient using elution A and B as shown in Table 18. The pH gradient during elution was achieved with the following linear gradient: elution A / elution B = ( 100: 0) -> (35:65) for 40 minutes. Table 18
[0344] [000344] The result of the No6 pH gradient elution is shown in Fig. 17. The homomeric antibody having the H chain of the anti-GPC3 antibody that was unable to bind to protein A passed through protein A; the first elution peak corresponded to the heteromeric antibody having the H chain of the anti-GPC3 antibody and the H chain of the anti-IL-6 receptor antibody; and the second elution peak corresponded to the homomeric antibody having the H chains of the anti-IL-6 receptor antibody. Thus, the present inventors demonstrated that by replacing Arg with His at position 435 (EU numbering), the heteromeric antibody having the anti-GPC3 antibody H chain and the anti-IL-6 receptor antibody H chain can be purified by step of purification based on protein A alone.
[0345] [000345] The first elution fraction was loaded onto a HiTrap SP Sepharose HP column (GE Healthcare) equilibrated with 20 mM sodium acetate buffer (pH 5.5). After washing with the same buffer, the column was eluted with a 0 to 500 mM NaCl concentration gradient. The resulting main peak was analyzed by cation exchange chromatography in the same manner as described above. The result is shown in Fig. 18. The bispecific antibody of interest has been shown to be purified to a very high purity. Industrial Applicability
[0346] [000346] The present invention provides efficient methods based on altering the binding capacity of protein A, to produce or purify high purity polypeptide multimers (multispecific antibodies) having the activity of binding two or more types of antigens by the purification step based on in protein A alone. Using the methods of the present invention, the polypeptide multimers of interest can be efficiently produced or purified to high purity without losing other effects produced by amino acid mutations of interest. Especially, when the methods are combined with a method to regulate the association between two types of protein domains, the polypeptide multimers of interest can be more efficiently produced or purified to a higher purity.

权利要求:
Claims (5)
[0001]
Method for producing a polypeptide multimer comprising a first polypeptide having antigen binding activity and a second polypeptide having antigen binding activity or no antigen binding activity, characterized by the fact that it comprises the following steps: (a) expressing DNA encoding the first polypeptide and DNA encoding the second polypeptide; and (b) collecting the expression product from step (a) using protein A affinity chromatography, wherein the first polypeptide and the second polypeptide comprise an amino acid sequence from an antibody Fc domain or an amino acid sequence from a constant region of the antibody heavy chain, wherein the antibody Fc domain or antibody heavy chain constant region is derived from human IgG; wherein the amino acid residue at position 435, according to EU numbering, in amino acid sequence of the Fc domain or the heavy chain constant region is histidine or arginine in the first polypeptide and arginine or histidine, respectively, in the second polypeptide; wherein the combination of amino acids at positions 356 and 439 in the amino acid sequence of the Fc region or constant region of the heavy chain of the first polypeptide has been modified to amino acids with the same electrical charge; and wherein the combination of amino acids at positions 356 and 439 in the amino acid sequence of the Fc region or constant region of the heavy chain of the second polypeptide has been modified to amino acids with an electrical charge opposite to that of positions 356 and 439 of the first polypeptide.
[0002]
Method according to claim 1, characterized by the fact that i) the purity of the collected polypeptide multimer is 95% or more; and / or ii) the first polypeptide having an antigen binding activity and the second polypeptide having an antigen binding activity comprise an amino acid sequence from a variable region of the antibody heavy chain, particularly, wherein at least one amino acid residue has been modified in the FR1, CDR2 and FR3 amino acid sequences of the variable region of the antibody heavy chain; and / or iii) wherein the polypeptide multimer comprises one or two third polypeptides that have antigen binding activity, and step (a) comprises the expression of DNA encoding the third polypeptide having antigen binding activity, particularly a) in which the third polypeptide having antigen-binding activity comprises an amino acid sequence of an antibody light chain, and / or b) wherein the polypeptide multimer additionally comprises a fourth polypeptide having an antigen binding activity, and step (a) comprises the expression of DNA encoding the fourth polypeptide having an antigen binding activity, especially b1) in which at least one of the third and fourth polypeptides that have antigen-binding activity comprises an amino acid sequence of an antibody light chain, or b2) wherein the first polypeptide having an antigen binding activity comprises amino acid sequences from a variable region of the antibody light chain and the constant region of the antibody heavy chain; the second polypeptide having antigen-binding activity comprises an amino acid sequence of an antibody heavy chain; the third polypeptide having antigen-binding activity comprises amino acid sequences from a variable region of the antibody heavy chain and a constant region from the antibody light chain; and the fourth polypeptide having antigen-binding activity comprises an amino acid sequence of an antibody light chain; and / or iv) where the polypeptide multimer is a multispecific antibody, particularly where the multispecific antibody is a bispecific antibody.
[0003]
Method according to any one of claims 1 and 2 i) to 2 iv), characterized in that it comprises the first polypeptide having an antigen binding activity and the second polypeptide having no antigen binding activity, and in that the first polypeptide having an antigen binding activity comprises an amino acid sequence of an antigen binding domain of a receptor and an amino acid sequence of an antibody Fc domain, and the second polypeptide which has no binding activity to the antigen comprises an amino acid sequence of an antibody Fc domain.
[0004]
Method for purifying a polypeptide multimer comprising a first polypeptide having antigen binding activity and a second polypeptide having antigen binding activity or no antigen binding activity, characterized by the fact that it comprises the following steps: (a) expressing the DNA encoding the first polypeptide and a DNA encoding the second polypeptide; and (b) collecting the expression product of step (a) by protein A affinity chromatography, wherein the first polypeptide and the second polypeptide comprise an amino acid sequence from an antibody Fc domain or an amino acid sequence from the region contained in the antibody heavy chain, wherein the antibody Fc domain or antibody heavy chain constant region is derived from human IgG; wherein the amino acid residue at position 435, according to EU numbering, in amino acid sequence of the Fc domain or the heavy chain constant region is histidine or arginine in the first polypeptide and arginine or histidine, respectively, in the second polypeptide; wherein the combination of amino acids at positions 356 and 439 in the amino acid sequence of the Fc region or constant region of the heavy chain of the first polypeptide has been modified to amino acids with the same electrical charge; and wherein the combination of amino acids at positions 356 and 439 in the amino acid sequence of the Fc region or constant region of the heavy chain of the second polypeptide has been modified to amino acids with an electrical charge opposite to that of positions 356 and 439 of the first polypeptide.
[0005]
Method according to claim 4, characterized by the fact that, i) the purity of the collected polypeptide multimer is 95% or more; and / or ii) the first polypeptide having an antigen binding activity and the second polypeptide having an antigen binding activity comprises an amino acid sequence from a variable region of the antibody heavy chain, particularly where at least one amino acid residue has been modified in the amino acid sequence of FR1, CDR2 and FR3 of the variable region of the antibody heavy chain; and / or iii) the polypeptide multimer comprises one or two third polypeptides that have antigen binding activity, and step (a) comprises the expression of the DNA encoding the third polypeptide having antigen binding activity, particularly a) in which the third polypeptide having antigen-binding activity comprises an amino acid sequence of an antibody light chain, and / or b) wherein the polypeptide multimer additionally comprises a fourth polypeptide having an antigen binding activity, and step (a) comprises the expression of the DNA encoding the fourth polypeptide having an antigen binding activity, especially b1) in which at least one of the third and fourth polypeptides that have antigen-binding activity comprises an amino acid sequence of an antibody light chain, or b2) wherein the first polypeptide having an antigen binding activity comprises amino acid sequences from an antibody light chain variable region and from the antibody heavy chain constant region; the second polypeptide having antigen-binding activity comprises an amino acid sequence of an antibody heavy chain; the third polypeptide having antigen-binding activity comprises an amino acid sequence of a variable region of the antibody heavy chain and a constant region of the antibody light chain; and the fourth polypeptide having antigen-binding activity comprises an amino acid sequence of an antibody light chain; and / or iv) the polypeptide multimer is a multispecific antibody, particularly, where the multispecific antibody is a bispecific antibody.

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WO2011078332A1|2011-06-30|

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US20200207805A1|2020-07-02|

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CA2785414A1|2011-06-30|

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法律状态:
2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-07-09| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NAO 10196/2001, QUE MODIFICOU A LEI NAO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUAANCIA PRA VIA DA ANVISA. CONSIDERANDO A APROVAA AO DOS TERMOS DO PARECER NAO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NAO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDAANCIAS CABA-VEIS. |

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2020-03-10| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2020-06-02| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-02-09| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-04-06| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 10 (DEZ) ANOS CONTADOS A PARTIR DE 06/04/2021, OBSERVADAS AS CONDICOES LEGAIS. |

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2021-05-25| B16C| Correction of notification of the grant [chapter 16.3 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 24/12/2010 OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF |

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2021-07-20| B09W| Correction of the decision to grant [chapter 9.1.4 patent gazette]|Free format text: RETIFICA-SE O DEFERIMENTO NOTIFICADO NA RPI NO 2614 DE 09/02/2021. |

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2021-08-31| B16C| Correction of notification of the grant [chapter 16.3 patent gazette]|Free format text: REFERENTE AO DESPACHO 16.1 PUBLICADO NA RPI 2622, QUANTO AOS DESENHOS |

优先权:

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申请号 | 申请日 | 专利标题

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JP2009294391|2009-12-25|

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JP2009-294391|2009-12-25|

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PCT/JP2010/073361|WO2011078332A1|2009-12-25|2010-12-24|Polypeptide modification method for purifying polypeptide multimers|

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