Multispecific antibodies and methods for producing the same

Abstract

The present invention provides a novel antibody format that does not use hetero-association technology and that is theoretically free of by-products having immune activity. This multispecific antibody has a Fab region that includes one polypeptide a chain and two polypeptide b chains. The polypeptide a chain includes a polypeptide in which a variable region Va1, a stationary region Ca1, a peptide linker LL, a variable region Va2 and a stationary region Ca are linked in the stated order. The polypeptide b chain includes a polypeptide in which a variable region Vb is linked to a stationary region Cb, which is linked to the stationary region Ca1 or the stationary region Ca2.

Description

[Technical Field]

[0001] The present invention relates to a multispecific antibody and a method for producing the same. [Background technology]

[0002] Bispecific antibodies are artificial antibodies that are highly functionalized by combining two types of antibodies to target two different antigens. The high functionality of bispecific antibodies results in distinctive mechanisms of action, such as immune cell recruitment, inhibition of receptor-mediated signaling, and mediation of protein-protein associations (Non-Patent Document 1).

[0003] Due to this unique mechanism of action, bispecific antibodies are expected to be a driving force in next-generation antibody drugs. For this reason, much research has been conducted on bispecific antibodies, and currently, more than 60 antibody formats have been reported (Non-Patent Document 2).

[0004] Bispecific antibodies are basically produced by introducing two heavy chain genes and two light chain genes into animal cells, expressing four polypeptide chains, and allowing them to naturally assemble. However, when the four expressed polypeptide chains assemble to form an antibody, each polypeptide chain is selected randomly, so unwanted antibodies, including homo-assembled antibodies, are inevitably produced as by-products. In this case, the theoretical yield of the desired antibody is 12.5%.

[0005] Therefore, in the production of bispecific antibodies, suppressing the production of by-products and improving the yield of the desired antibody are important issues. To address this issue, vigorous research is being conducted into methods for promoting hetero-association of polypeptide chains. In such hetero-association techniques, various mutations have been proposed, including methods for introducing substitutions into the CH1 domain and CL domain (Patent Document 1) and methods for introducing mutations into the CH3 domain (Non-Patent Document 3, Patent Document 2). Thanks to the accumulation of numerous hetero-association techniques, the yield of bispecific antibodies produced by hetero-association has reached over 90% (Non-Patent Document 4).

[0006] On the other hand, it has been pointed out that by-products generated during the manufacturing process of bispecific antibodies may cause unexpected activation of immune cells, increasing the risk of side effects when used as antibody drugs (Non-Patent Document 5). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Drug Discovery Today 20, 838?847 (2015) [Non-patent document 2] Molecular Immunology 67, 95?106 (2015) [Non-patent document 3] HFront. Immunol. 7, 394 (2016) [Non-patent document 4] MAbs, 9, 182-212 (2017) [Non-Patent Document 5] Bispecific Antibody Development Programs: Guidance for Industry, US Food and Drug Administration (2019) [Patent documents]

[0008] [Patent Document 1] International Publication No. 2006 / 106905 [Patent Document 2] International Publication No. 2013 / 157953 Summary of the Invention [Problem to be solved by the invention]

[0009] Heterogeneous association technology, which has undergone repeated improvements in the production of bispecific antibodies, can be considered a sophisticated technology in light of the significant contributions it has made to improving yields. However, no matter how much heterogeneous association technology is improved, it is theoretically inevitable that homogeneous association antibodies will be produced as by-products. Homogeneous association antibodies, even at a fraction of a few percent, can induce unwanted immune responses and increase the risk of side effects. Furthermore, removing such homogeneous association antibodies requires specialized and complex separation and purification processes. Considering these problems, it must be said that methods for producing bispecific antibodies based on heterogeneous association have inherent limitations.

[0010] Therefore, the present inventors have shifted the conventional paradigm based on hetero-association technology in the production of bispecific antibodies and focused on an approach that theoretically does not involve by-products exhibiting immunoreactivity.

[0011] In other words, the present invention aims to provide a new antibody format that does not use hetero-association technology and is theoretically free from immunoreactive by-products. [Means for solving the problem]

[0012] As a result of extensive research, the present inventors have found that by designing a bispecific antibody so that the variable regions of both arms are present on the same polypeptide chain by linking the constant region of one arm with a peptide linker, the bispecific antibody will consist of only two types of polypeptide chains, and therefore will theoretically not produce immunoreactive by-products. The present invention was completed based on this finding. Specifically, the present invention provides the following aspects.

[0013] Item 1. A multispecific antibody having a Fab region comprising one polypeptide a chain and two polypeptide b chains: A polypeptide a chain comprising a polypeptide in which a variable region Va1, a constant region Ca1, a peptide linker LL, a variable region Va2, and a constant region Ca2 are linked in this order; and A polypeptide b chain comprising a polypeptide in which a variable region Vb is linked to a constant region Cb that binds to the constant region Cali or the constant region Ca2. Item 2. The multispecific antibody according to Item 1, wherein the length of the peptide linker LL is 70 to 280 Å. Item 3. The multispecific antibody according to Item 1 or 2, wherein the peptide linker LL comprises a protease recognition sequence. Item 4. The multispecific antibody according to Item 3, wherein the peptide linker LL comprises a protease recognition sequence Lr1 on the constant region Ca1 side and a protease recognition sequence Lr2 on the variable region Va2 side. Item 5. The multispecific antibody according to any one of Items 1 to 4, which is IgD, IgE, IgG, or F(ab')2. Item 6. The multispecific antibody according to any one of Items 1 to 5, wherein the polypeptide a chain comprises a polypeptide in which a heavy chain variable region VHa1, a heavy chain constant region CHa1, a peptide linker LL, a heavy chain variable region VHa2, and a heavy chain constant region CHa2 are linked in this order. Item 7. The multispecific antibody according to any one of Items 1 to 5, wherein the polypeptide a chain comprises a polypeptide in which a heavy chain variable region VHa1, a light chain constant region CLa1, a peptide linker LL, a heavy chain variable region VHa2, and a light chain constant region CLa2 are linked in this order. Item 8. The multispecific antibody according to any one of Items 1 to 7, wherein a single-chain antibody is further bound to the variable region Va1 and / or the constant region Ca2. Item 9. A multispecific antibody having a Fab region comprising one polypeptide a' chain described below, one polypeptide a'' chain described below, and two polypeptide b chains described below: a polypeptide a' chain comprising a polypeptide in which a variable region Va1, a constant region Ca1, and a cleavage fragment Lr1' of a protease recognition sequence Lr1 are linked in this order; a polypeptide a'' chain comprising a polypeptide in which a cleavage fragment Lr2' of the protease recognition sequence Lr2, a variable region Va2, and a constant region Ca2 are linked in this order; and A polypeptide b chain comprising a polypeptide in which a variable region Vb is linked to a constant region Cb that binds to the constant region Cali or the constant region Ca2. Item 10. DNA encoding a polypeptide a chain comprising a polypeptide in which a variable region Va1, a constant region Ca1, a peptide linker LL, a variable region Va2, and a constant region Ca2 are linked in this order, the DNA being used to produce the multispecific antibody of any of Items 1 to 9. Item 11. A recombinant vector va containing DNA encoding a polypeptide a chain comprising a polypeptide in which a variable region Va1, a constant region Ca1, a peptide linker LL, a variable region Va2, and a constant region Ca2 are linked in this order; a recombinant vector vb containing DNA encoding a polypeptide b chain comprising a polypeptide in which a variable region Vb and a constant region Cb that binds to the constant region Cali or the constant region Ca2 are linked; A transformant obtained by transforming a host with the Item 12. A recombinant vector va containing DNA encoding a polypeptide a chain comprising a polypeptide in which a variable region Va1, a constant region Ca1, a peptide linker LL, a variable region Va2, and a constant region Ca2 are linked in this order; a recombinant vector vb containing DNA encoding a polypeptide b chain comprising a polypeptide in which a variable region Vb and a constant region Cb that binds to the constant region Cali or the constant region Ca2 are linked; A method for producing a multispecific antibody, comprising an antibody production step of transforming a host with the above-mentioned vector and culturing the resulting transformant. Item 13. The peptide linker LL comprises a protease recognition sequence Lr1 on the constant region Ca1 side and a protease recognition sequence Lr2 on the variable region Va2 side; Aspect 13. The method for producing a multispecific antibody according to Aspect 12, further comprising, after the antibody production step, a linker cleavage step of cleaving the peptide linker LL in the produced antibodies using a protease corresponding to the protease recognition sequence Lr1 and the protease recognition sequence Lr2. Item 14. An expression vector va' comprising, in this order, a cloning site CS1 for incorporating the variable region Va1, DNA encoding the constant region Ca1, DNA encoding the peptide linker LL, a cloning site CS2 for incorporating the variable region Va2, and DNA encoding the constant region Ca2; an expression vector vb' containing a cloning site CS for inserting a variable region Vb and DNA encoding a constant region Cb that binds to the constant region Cali or the constant region Ca2; A kit for producing a multispecific antibody, comprising: Item 15. A diagnostic agent comprising the multispecific antibody according to any one of Items 1 to 9. Item 16. A pharmaceutical composition comprising the multispecific antibody according to any one of Items 1 to 9. [Effects of the Invention]

[0014] The present invention provides a new antibody format that does not use hetero-association techniques and is theoretically free of immunoreactive by-products. [Brief explanation of the drawings]

[0015] [Figure 1] Among the multispecific antibodies of the present invention, an example of an IgD,E,G bispecific antibody is shown schematically. [Figure 2] The polypeptide chains that make up the multispecific antibody in FIG. 1 are shown schematically. [Figure 3] FIG. 1 shows a schematic diagram of a specific example of a multispecific antibody. [Figure 4] Among the multispecific antibodies of the present invention, an example of a F(ab')-type bispecific antibody is shown schematically. [Figure 5] Another specific example of the multispecific antibody of FIG. 1 is shown schematically. [Figure 6]FIG. 5 shows a schematic diagram of by-products generated in the production of a multispecific antibody. [Figure 7] Another specific example of the multispecific antibody of FIG. 1 is shown schematically. [Figure 8] Among the multispecific antibodies of the present invention, an example of a trispecific antibody is shown schematically. [Figure 9] Another example of a trispecific antibody among the multispecific antibodies of the present invention is shown schematically. [Figure 10] Among the multispecific antibodies of the present invention, an example of a tetraspecific antibody is shown schematically. [Figure 11] Among the multispecific antibodies of the present invention, examples of bispecific antibodies in which the binding valency for one of the epitopes is increased are shown schematically. [Figure 12] Another example of a bispecific antibody of the multispecific antibody of the present invention, in which the binding valency for one of the epitopes is increased, is shown schematically. [Figure 13] Among the multispecific antibodies of the present invention, an example of a trispecific antibody with increased binding valency to an epitope is shown schematically. [Figure 14] Another example of the multispecific antibodies of the present invention, an IgD,E,G bispecific antibody, is shown schematically. [Figure 15] 1 shows a schematic diagram of a recombinant vector of the present invention. [Figure 16] Another example of a multispecific antibody obtained by the method for producing a multispecific antibody of the present invention is schematically shown. [Figure 17] 1 is a schematic diagram showing an expression vector included in the kit of the present invention. [Figure 18] 1 is a schematic diagram showing the anti-HER2×HER3 bispecific antibody designed and prepared in Test Example 1. [Figure 19] 1 is a schematic diagram showing the recombinant vector used in Test Example 1 for expressing the anti-HER2×HER3 bispecific antibody of interest. [Figure 20] 1 shows an electrophoresis diagram obtained in Test Example 1 under reducing conditions for a Protein A affinity purified product of an anti-HER2×HER3 bispecific antibody (Examples 1 to 5). [Figure 21]1 shows an electrophoresis diagram obtained in Test Example 1 under non-reducing conditions for a Protein A affinity purified product of an anti-HER2×HER3 bispecific antibody (Examples 1 to 5). [Figure 22] 1 shows a gel filtration chromatogram of the Protein A affinity purified product of the anti-HER2×HER3 bispecific antibody (Example 1) product obtained in Test Example 1. [Figure 23] 1 shows an electrophoresis diagram obtained in Test Example 1 under non-reducing conditions for the Protein A affinity purified product of the anti-HER2×HER3 bispecific antibody (Example 1). [Figure 24] 1 shows a gel filtration chromatogram of a Protein A affinity purified product of an anti-HER2×HER3 bispecific antibody product (Examples 2 to 4) obtained in Test Example 1. [Figure 25] 1 shows a gel filtration chromatogram of the Protein A affinity purified product of the anti-HER2×HER3 bispecific antibody (Example 5) product obtained in Test Example 1. [Figure 26] 1 shows an electrophoresis diagram under non-reducing conditions of a fraction obtained in Test Example 1 by purifying the Protein A affinity-purified product of the anti-HER2×HER3 bispecific antibody (Example 1) using an IgG-CH1-binding carrier. [Figure 27] 1 shows a cation exchange chromatogram of the Protein A affinity purified product of the anti-HER2×HER3 bispecific antibody (Example 1) obtained in Test Example 1. [Figure 28] 1 shows the results obtained in Test Example 2 of evaluating the binding activity of anti-HER2×HER3 bispecific antibodies (Examples 1 to 4) to MCF-7 by flow cytometry. [Figure 29] 1 shows the results obtained in Test Example 2 of evaluating the bispecificity of the anti-HER2×HER3 bispecific antibody (Example 1) by SPR method. [Figure 30] The bispecificity of the anti-HER2×HER3 bispecific antibodies (Examples 1 to 5) obtained in Test Example 2 to MCF-7 is shown by the results of evaluating their cell proliferation inhibitory ability. [Figure 31]1 is a schematic diagram showing the anti-HER2×HER3 bispecific antibody designed and prepared in Test Example 4. [Figure 32] 1 is a schematic diagram showing the recombinant vector used in Test Example 4 for expressing the anti-HER2×HER3 bispecific antibody of interest. [Figure 33] 1 shows an electrophoresis diagram of the Protein A affinity purified product of the anti-HER2×HER3 bispecific antibody (Example 9) obtained in Test Example 4. [Figure 34] 1 shows a gel filtration chromatogram of the Protein A affinity purified product of the anti-HER2×HER3 bispecific antibody (Example 9) product obtained in Test Example 4. [Figure 35A] 1 shows a schematic diagram of the anti-CD20×CD3 bispecific antibody (Examples 10 and 11) designed and prepared in Test Example 5. [Figure 35B] 1 shows a schematic diagram of the anti-BCMA×CD3 bispecific antibody (Examples 12 and 13) designed and prepared in Test Example 5. [Figure 36A] 1 is a schematic diagram showing the recombinant vector used in Test Example 5 for expressing the anti-CD20×CD3 bispecific antibody of interest (Examples 10 and 11). [Figure 36B] FIG. 1 shows a schematic diagram of the recombinant vector used in Test Example 5 to express the anti-BCMA×CD3 bispecific antibody of interest (Examples 12 and 13). [Figure 37] 1 shows cation exchange chromatograms of Protein A affinity purified products of anti-CD20×CD3 bispecific antibody (Examples 10 and 11) and anti-BCMA×CD3 bispecific antibody (Examples 12 and 13) products obtained in Test Example 5. [Figure 38] FIG. 1 shows electropherograms of the peaks separated by cation exchange chromatography of the anti-CD20×CD3 bispecific antibody (Examples 10 and 11) products and the anti-BCMA×CD3 bispecific antibody (Examples 12 and 13) products obtained in Test Example 5. [Figure 39] 1 shows the results of flow cytometry analysis obtained in Test Example 6 to evaluate the binding activity of anti-CD20×CD3 bispecific antibodies (Examples 10 and 11) to CD20-positive cells and CD3-positive cells. [Figure 40A] 1 shows the results of flow cytometry analysis obtained in Test Example 6 to evaluate the binding activity of anti-BCMA×CD3 bispecific antibodies (Examples 12 and 13) to CD3-positive cells. [Figure 40B] 1 shows the results of surface plasmon resonance analysis obtained in Test Example 6 to evaluate the binding activity of anti-BCMA×CD3 bispecific antibodies (Examples 12 and 13) to BCMA. [Figure 41A] 1 shows the results of a toxicity test using CD20-positive cells and CD3-positive cells, obtained in Test Example 6, to evaluate the bispecificity of the anti-BCMA×CD3 bispecific antibody (Example 10). [Figure 41B] 1 shows the results of flow cytometry analysis of BCMA- and CD3-positive cells obtained in Test Example 6, evaluating the bispecificity of anti-BCMA×CD3 bispecific antibodies (Examples 12 and 13). [Figure 42] 1 shows an electrophoresis diagram of a linker-cleaved product (Example 14) of the anti-HER2×HER3 bispecific antibody of Example 1, obtained in Test Example 7. [Figure 43] 1 shows the results of flow cytometry analysis obtained in Test Example 7, evaluating the binding activity of the linker-cleaved product of the anti-HER2×HER3 bispecific antibody of Example 1 (Example 14) to HER2- and HER3-positive cells. DETAILED DESCRIPTION OF THE INVENTION

[0016] 1. Multispecific antibodies The multispecific antibodies of the present invention are characterized by having a Fab region comprising one predetermined polypeptide a chain and two predetermined polypeptide b chains. The predetermined polypeptide a chain comprises a polypeptide in which a variable region Va1, a constant region Ca1, a peptide linker LL, a variable region Va2, and a constant region Ca2 are linked in this order. The predetermined polypeptide b chain comprises a polypeptide in which a variable region Vb is linked to a constant region Cb that binds to the constant region Ca1 or the constant region Ca2.

[0017] 1-1. Multispecific antibody 1 Figure 1 schematically shows an example of an IgD,E,G bispecific antibody (multispecific antibody 1) as a representative multispecific antibody of the present invention. The polypeptide a chain constituting multispecific antibody 1 in Figure 1 is composed of a variable region Va1, a constant region Ca1, a peptide linker LL, a variable region Va2, and a constant region Ca2 linked in this order. The polypeptide b chains each contain a variable region Vb and a constant region Cb, and in this example also contain constant regions CH2 and CH3.

[0018] Figure 2 shows a schematic representation of the polypeptide chains that make up multispecific antibody 1 in Figure 1. Typically, the Fab region of an antibody is composed of four polypeptide chains, but in multispecific antibody 1 of the present invention, of the four polypeptide chains that normally make up the Fab region, two are linked by a peptide linker LL to form a single polypeptide a chain, and the remaining two polypeptide chains are shared as a polypeptide b chain. Because the polypeptide chains that make up the antibody are simplified to just two types, the polypeptide a chain and the polypeptide b chain, multispecific antibody 1 of the present invention has a format that theoretically does not produce immunoreactive by-products when the polypeptide a chain and the polypeptide b chain are associated with each other in any combination.

[0019] A multispecific antibody is defined as an antibody having specificity for two or more different epitopes; in other words, an antibody containing two or more variable regions (so-called Fvs) composed of a heavy chain variable region (so-called VH) and a light chain variable region (so-called VL). In the bispecific antibody multispecific antibody 1 shown in FIG. 1, variable region Va1 and variable region Vb, and variable region Va2 and variable region Vb constitute two types of Fvs (areas enclosed by dashed lines in the figure) that have specificity for different epitopes. The sequences of variable region Va1 and variable region Va2 need only be different from each other, and the specific sequences of variable region Va1 and variable region Va2 and the variable regions are appropriately selected so that the Fvs constituted by them have different complementarity-determining regions (so-called CDRs). Furthermore, the specific sequences of the CDRs are selected without limitation depending on the epitope targeted by multispecific antibody 1.

[0020] The sequences of the constant region CH2 and constant region CH3 are also appropriately selected from those that constitute the Fc region. The Fc region is a region that is cleaved by papain enzyme and is known to be involved in complement activation, C1q binding, C3 activation, and Fc reporter binding. The specific sequence of the Fc region is selected without limitation depending on the antibody isotype (IgD, IgE, IgG).

[0021] The bond between the constant region Cb of the polypeptide b chain and the constant region Ca1 of the polypeptide a chain, the bond between the constant region Cb of the polypeptide b chain and the constant region Ca2 of the polypeptide a chain, and the bond between the two polypeptide b chains are usually disulfide bonds.

[0022] In the polypeptide a chain, the length of the peptide linker LL linking the constant region Ca1 and the variable region Va2 is not particularly limited, and examples include 70 to 280 Å or 20 to 80 amino acid residues.

[0023] From the viewpoint of further suppressing the production of by-products, the lower limit of the range of the length of the peptide linker LL is preferably 122 Å or more, more preferably 140 Å or more, even more preferably 175 Å or more, still more preferably 210 Å or more, even more preferably 227 Å or more, and particularly preferably 234 Å or more. Alternatively, from the viewpoint of further suppressing the production of by-products, the lower limit of the range of the length of the peptide linker LL is preferably 35 amino acid residues or more, more preferably 40 amino acid residues or more, even more preferably 50 amino acid residues or more, still more preferably 60 amino acid residues or more, even more preferably 65 amino acid residues or more, and particularly preferably 67 amino acid residues or more.

[0024] The upper limit of the length range of the peptide linker LL is preferably 26 Å or less, more preferably 252 Å or less, and even more preferably 245 Å or less. Furthermore, when a multispecific antibody is used at a low concentration and / or when multiple epitopes targeted by the multispecific antibody are close to each other, in order to enhance binding activity, the upper limit of the length range of the peptide linker LL is preferably 210 Å or less, more preferably 192 Å or less, even more preferably 140 Å or less, even more preferably 122 Å or less, still more preferably 105 Å or less, and particularly preferably 87 Å or less. Alternatively, the upper limit of the length range of the peptide linker LL is preferably 75 amino acid residues or less, more preferably 72 amino acid residues or less, and even more preferably 70 amino acid residues or less. Furthermore, when multispecific antibodies are used at low concentrations and / or when multiple epitopes targeted by the multispecific antibodies are located close to each other, the upper limit of the length range of the peptide linker LL, from the viewpoint of enhancing binding activity, is preferably 60 amino acid residues or less, more preferably 55 amino acid residues or less, even more preferably 40 amino acid residues or less, even more preferably 35 amino acid residues or less, even more preferably 30 amino acid residues or less, and particularly preferably 25 amino acid residues or less. Specific examples of the low concentration include, for example, 2 to 50 nM, preferably 4 to 30 nM, more preferably 6 to 20 nM, and even more preferably 8 to 15 nM. A specific example of a case where multiple epitopes targeted by the multispecific antibody are located close to each other is when multiple antigens (e.g., two antigens) form a heteromultimer (e.g., a heterodimer) on the surface of a single cell.

[0025] The specific sequence of the peptide linker LL is appropriately selected so that it does not cause undesired binding during expression of the polypeptide a chain or during association between the polypeptide a chain and the polypeptide b chain, and does not affect the molecular recognition ability of the multispecific antibody 1 itself.

[0026] The peptide linker LL mainly comprises a basic sequence that contributes to linkage. The amino acid residues constituting the basic sequence of the peptide linker LL preferably include amino acid residues that do not have bulky side chains or side chains at all. Such amino acid residues preferably include glycine residues, alanine residues, serine residues, threonine residues, aspartic acid residues, glutamic acid residues, etc., and more preferably include glycine residues and serine residues.

[0027] Furthermore, the basic sequence of the peptide linker LL preferably contains hydrophilic amino acid residues. Examples of such hydrophilic amino acid residues include serine, threonine, aspartic acid, and glutamic acid residues, with serine residues being preferred. In this case, examples of hydrophobic amino acid residues other than the hydrophilic amino acid residues constituting the basic sequence of the peptide linker LL include glycine and alanine residues, with glycine residues being preferred. The proportion of hydrophilic amino acid residues in the total number of amino acid residues constituting the peptide linker LL is preferably 10 to 30%, more preferably 15 to 25%, and even more preferably 18 to 22%. A preferred basic sequence of the peptide linker LL is one in which hydrophobic and hydrophilic amino acid residues are alternately repeated, with glycine (G) and serine (S) residues being more preferred, and with GGGGS repeats being particularly preferred.

[0028] 1-2. Multispecific antibody 11 In the multispecific antibodies of the present invention, the peptide linker LL may consist of only the basic sequence (without containing a protease recognition sequence), or may further contain a protease recognition sequence in addition to the basic sequence. From the viewpoint of suppressing the production of by-products in the production of the multispecific antibodies of the present invention, a peptide linker LL that does not contain a protease recognition sequence is preferred. On the other hand, from the viewpoint of performing processing to cleave the peptide linker LL after antibody production, a peptide linker LL that contains a protease recognition sequence is used.

[0029] When the peptide linker LL contains a protease recognition sequence, the number of protease recognition sequences per peptide linker LL is not particularly limited and may be, for example, 1 or 2 or more, preferably 1 or 2, and more preferably 2. Furthermore, when the peptide linker LL contains two or more protease recognition sequences, the two or more protease recognition sequences may be the same or different from each other. Furthermore, the protease recognition sequence is not particularly limited as long as it is a sequence that is specifically recognized and cleaved by a specific protease.

[0030] Another example of an IgD,E,G bispecific antibody (multispecific antibody 11) in which the peptide linker LL in the multispecific antibody of the present invention contains a protease recognition sequence is shown schematically in Figure 3. Multispecific antibody 11 in Figure 3 is similar to multispecific antibody 1 in Figure 1, except that the peptide linker L in the multispecific antibody of Figure 1 is replaced with peptide linker LL-11 containing two protease recognition sequences Lr1 and Lr2, and the polypeptide a chain in the multispecific antibody of Figure 1 is replaced with polypeptide a-11 chain containing peptide linker LL-11.

[0031] Specifically, the peptide linker LL-11 in multispecific antibody 11 comprises a protease recognition sequence Lr1 on the constant region Ca1 side and a protease recognition sequence Lr2 on the variable region Va2 side. The protease recognition sequence Lr1 is preferably located as close as possible to the constant region Ca1, and more preferably adjacent to the constant region Ca1. The protease recognition sequence Lr2 is preferably located as close as possible to the variable region Va2, and more preferably adjacent to the variable region Va2.

[0032] 1-3. Multispecific antibody 1' In the present invention, multispecific antibodies are not particularly limited in terms of the shape of other portions, as long as they have a Fab region containing one predetermined polypeptide a chain and two predetermined polypeptide b chains. For example, an antibody Fc region is not necessarily required.

[0033] An example of a F(ab')-type bispecific antibody (multispecific antibody 1') from the multiple antibodies of the present invention is shown schematically in Figure 4. Multispecific antibody 1' shown in Figure 4 is similar to multispecific antibody 1 shown in Figure 1, except that the polypeptide b chain does not have CH2 and CH3 in the constant region.

[0034] 1-4. Multispecific antibody 12 In the above-mentioned multispecific antibodies 1, 11, and 1', the specific sequences of the variable region Va1, constant region Ca1, variable region Va2, and constant region Ca2 of the polypeptide a and a-11 chains, and the variable region Vb and constant region Cb of the polypeptide b chain may each independently be the sequences of either the heavy chain or the light chain.

[0035] Figure 5 schematically shows a specific example of an IgD,E,G bispecific antibody (multispecific antibody 12) among the multispecific antibodies of the present invention. Multispecific antibody 12 in Figure 5 is an example of multispecific antibody 1 in Figure 1, and specifically illustrates a case in which the polypeptide a chain and polypeptide b chain in multispecific antibody 1 in Figure 1 are polypeptide a-12 chain and polypeptide b-12 chain, respectively.

[0036] In this polypeptide a-12 chain, the variable region Va1, constant region Ca1, variable region Va2, and constant region Ca2 of the polypeptide a chain in multispecific antibody 1 in Figure 1 all have the heavy chain sequence; that is, as shown in Figure 5, the heavy chain variable region VHa1, heavy chain constant region CHa1, peptide linker LL, heavy chain variable region VHa2, and heavy chain constant region CHa2 are linked in this order.

[0037] Furthermore, in the polypeptide b-12 chain, the variable region Vb and constant region Cb of the polypeptide b chain in multispecific antibody 1 in Figure 1 both have light chain sequences; that is, the light chain variable region VLb and light chain constant region CLb are linked as shown in Figure 5. The polypeptide b-12 chain has a non-natural domain junction in which the light chain constant region CLb is further linked to the constant region CH2 and constant region CH3. Methods for determining the amino acid sequence of such non-natural domain junctions are known, and those skilled in the art would be able to appropriately determine the amino acid sequence of the junction when designing multispecific antibody 12.

[0038] As described above, the multispecific antibodies of the present invention theoretically do not produce immunoreactive by-products. For example, when the polypeptide a-12 chain and polypeptide b-12 chain constituting multispecific antibody 12 shown in Figure 5 associate, the only immunoreactive product produced by any combination of these polypeptide chains is, in theory, multispecific antibody 12. On the other hand, an example of a by-product produced when the polypeptide a-12 chain and polypeptide b-12 chain constituting multispecific antibody 12 associate is the polypeptide b-12 chain tetramer 12BQ shown in Figure 6. Although this by-product is produced together with multispecific antibody 12, it does not exhibit immunoreactivity. Therefore, unlike previous multispecific antibodies that inevitably produce immunoreactive by-products, the present invention does not require a special and complicated separation process and can also avoid undesired immune reactions caused by small amounts of by-products remaining after the separation process.

[0039] As a further modification of the multispecific antibody 12, the peptide linker LL may be replaced with a peptide linker LL-11 containing a protease recognition sequence, or the multispecific antibody 12 may be an F(ab') type lacking the Fc region.

[0040] 1-5. Multispecific antibodies 13 Furthermore, Figure 7 schematically shows another specific example of an IgD,E,G bispecific antibody (multispecific antibody 13) among the multispecific antibodies of the present invention. Multispecific antibody 13 in Figure 7 is another example of multispecific antibody 1 in Figure 1, and specifically illustrates a case in which the polypeptide a chain and polypeptide b chain in multispecific antibody 1 in Figure 1 are polypeptide a-13 chain and polypeptide b-13 chain, respectively.

[0041] In this polypeptide a-13 chain, the variable regions Va1 and Va2 of the polypeptide a chain in multispecific antibody 1 in Figure 1 have heavy chain sequences, and the constant regions Ca1 and Ca2 have light chain sequences; that is, as shown in Figure 7, the heavy chain variable region VHa1, light chain constant region CLa1, peptide linker LL, heavy chain variable region VHa2, and light chain constant region CLa2 are linked in this order.

[0042] Furthermore, in the polypeptide b-13 chain, the variable region Vb of the polypeptide b chain in multispecific antibody 1 in Figure 1 has a light chain sequence, and the constant region Cb has a heavy chain sequence; that is, as shown in Figure 7, the light chain variable region VLb and the light chain constant region CHb are linked.

[0043] The polypeptide a-13 chain and the polypeptide b-13 chain have a non-natural domain junction in which a light chain-derived region is linked to a heavy chain-derived region. Methods for determining the amino acid sequence of such non-natural domain junctions are known, and those skilled in the art can appropriately determine the amino acid sequence of the junction when designing multispecific antibody 13.

[0044] As mentioned above, when the polypeptide a-13 chain and the polypeptide b-13 chain associate with each other in any combination of polypeptide chains, the only immunoreactive product produced in the case of multispecific antibody 13 is, in theory, multispecific antibody 13. However, no tetramer corresponding to tetramer 12BQ in Figure 6 is produced as a by-product. For this reason, the format of multispecific antibody 13 is even more preferable in that it further suppresses the production of by-products and enables higher yields.

[0045] As a further modification of the multispecific antibody 13, the peptide linker LL may be replaced with a peptide linker LL-11 containing a protease recognition sequence, or the multispecific antibody 13 may be an F(ab') type lacking the Fc region.

[0046] 1-6. Multispecific antibodies21,22,23 The multispecific antibodies of the present invention can be designed so that a single-chain antibody further binds to the variable region Va1 and / or the constant region Ca2.

[0047] A single-chain antibody is a well-known structure consisting of a heavy chain variable region (so-called VH), a light chain variable region (so-called VL), and a peptide linker connecting them, constituting a variable region (so-called Fv) composed of VH and VL.

[0048] As examples of multispecific antibodies of the present invention to which single-chain antibodies are bound, specific examples of trispecific antibodies (multispecific antibodies 21 and 22) and a specific example of tetraspecific antibody (multispecific antibody 23) are schematically shown in Figures 8 to 10.

[0049] Multispecific antibody 21 shown in Figure 8 is similar to multispecific antibody 1 in Figure 1, except that the polypeptide a chain in the multiple antibody in Figure 1 is replaced by a polypeptide a-21 chain in which a single-chain antibody ScFv1 is bound to the variable region Va1. Multispecific antibody 22 shown in Figure 9 is similar to multispecific antibody 1 in Figure 1, except that the polypeptide a chain in the multiple antibody in Figure 1 is replaced by a polypeptide a-22 chain in which a single-chain antibody ScFv2 is bound to the constant region Ca2. Multispecific antibody 23 in Figure 10 is similar to multispecific antibody 1 in Figure 1, except that the polypeptide a chain in the multiple antibody in Figure 1 is replaced by a polypeptide a-23 chain in which a single-chain antibody ScFv1 is bound to the variable region Va1 and a single-chain antibody ScFv2 is bound to the constant region Ca2.

[0050] In multispecific antibodies 21, 22, and 23, the Fvs composed of VH and VL are shown enclosed in dashed lines, and these Fvs are all constructed to have specificity for different epitopes. Accordingly, in multispecific antibodies 21 and 22, the specific sequences of VH and VL of single-chain antibodies ScFv1 and 2 are appropriately selected so that the complementarity-determining regions (CDRs) of the Fvs they constitute are different from the CDRs of the Fvs composed of variable regions Va1 and Vb and the CDRs of the Fvs composed of variable regions Va2 and Vb. Furthermore, in multispecific antibody 23, the VH and VL of single-chain antibody ScFv1 and the VH and VL of single-chain antibody ScFv2 are selected so that they constitute different CDRs.

[0051] In the polypeptide a-21 chain of multispecific antibody 21, either the VH or VL of single-chain antibody ScFv1 may be linked to the variable region Va1. Similarly, in the polypeptide a-22 chain of multispecific antibody 22, either the VH or VL of single-chain antibody ScFv2 may be linked to the constant region Ca2. Similarly, in the polypeptide a-23 chain of multispecific antibody 23, either the VH or VL of single-chain antibody ScFv1 may be linked to the variable region Va1, and either the VH or VL of the independent single-chain antibody ScFv2 may be linked to the constant region Ca2.

[0052] The sequence and length of the peptide linker linking the VH and VL that constitute the single-chain antibody ScFv1,2 can be appropriately selected by those skilled in the art, taking into consideration the stability, three-dimensional structure formation, antigen recognition ability, etc. of the single-chain antibody ScFv. Specific constituent amino acids and sequences are the same as the basic sequence described above for the peptide linker LL, and more specific examples include GGGGS or its repeats. Specific lengths include approximately 15 amino acid residues.

[0053] The linking mode between the single-chain antibody ScFv1, 2 and the variable region Va1 or the constant region Ca2 is not particularly limited, but is preferably via a peptide linker. The sequence and length of the peptide linker linking the single-chain antibody ScFv1, 2 and the variable region Va1 or the constant region Ca2 can be appropriately selected by those skilled in the art, taking into consideration the size and shape of the antigen, the relative positions of different epitopes, and the like. Specific constituent amino acids and sequences are the same as the basic sequence described above for the peptide linker LL, and more specific examples include GGGGS or its repeats. Specific lengths include, for example, 5 to 20 amino acid residues, preferably 8 to 15 amino acid residues, and more preferably about 10 to 12 amino acid residues.

[0054] The specific sequences of the variable region Va1, constant region Ca1, variable region Va2, and constant region Ca2 in the polypeptide a-21 chain, polypeptide a-22 chain, and polypeptide a-23 chain constituting each of multispecific antibodies 21 to 23, and the variable region Vb and constant region Cb in the polypeptide b chain, may each independently be the sequences of either the heavy chain or the light chain.

[0055] Therefore, in one example of multispecific antibodies 21 to 23, the variable region Va1, constant region Ca1, variable region Va2, and constant region Ca2 in the polypeptide a-21 chain, polypeptide a-22 chain, and polypeptide a-23 chain may all have heavy chain sequences, and the variable region Vb and constant region Cb of the polypeptide b chain may all have light chain sequences, similar to multispecific antibody 12 in Figure 5. Furthermore, in another example of multispecific antibodies 21 to 23, the variable region Va1 and variable region Va2 in the polypeptide a-21 chain, polypeptide a-22 chain, and polypeptide a-23 chain may all have heavy chain sequences, and the constant regions Ca1 and Ca2 have light chain sequences, similar to multispecific antibody 13 in Figure 7. In addition, in the polypeptide b chain, the variable region Vb may have a light chain sequence and the constant region Cb may have a heavy chain sequence.

[0056] As further variations of multispecific antibodies 21 to 23, the peptide linker LL may be replaced with peptide linker LL-11 containing a protease recognition sequence, or the Fc region may be absent.

[0057] 1-7. Multispecific antibodies31,32,33 The multispecific antibodies of the present invention can also be designed as multispecific antibodies with increased valency for at least one epitope, in addition to those described above in "1-6. Multispecific antibodies 21, 22, and 23," by designing the variable region Va1 and / or the constant region Ca2 to further bind to a single-chain antibody.

[0058] 11 and 12 schematically show specific examples of (2+1) type bispecific antibodies (multispecific antibodies 31 and 32) among the multispecific antibodies of the present invention, and FIG. 13 schematically shows a specific example of a (2+1+1) type trispecific antibody (multispecific antibody 33) among the multispecific antibodies of the present invention.

[0059] Multispecific antibody 31 shown in Figure 11 is similar to multispecific antibody 1 in Figure 1, except that the polypeptide a-31 chain replaces the polypeptide a chain in the multispecific antibody of Figure 1. The polypeptide a-31 chain is designed so that single-chain antibody ScFv1 binds to variable region Va1, and the CDRs of the Fv consisting of variable region Va2 and variable region Vb are shared with the CDRs of the Fv consisting of variable region Va1 and variable region Vb but are different from the CDRs of single-chain antibody ScFv1.

[0060] Multispecific antibody 32 shown in Figure 12 is similar to multispecific antibody 1 in Figure 1, except that the polypeptide a-32 chain replaces the polypeptide a chain in the multispecific antibody in Figure 1. The polypeptide a-32 chain is designed so that the single-chain antibody ScFv2 binds to the constant region Ca2, and the CDRs of the Fv consisting of the variable regions Va2 and Vb are shared with the CDRs of the Fv consisting of the variable regions Va1 and Vb but are different from the CDRs of the single-chain antibody ScFv2.

[0061] Multispecific antibody 33 shown in Figure 13 is similar to multispecific antibody 1 in Figure 1, except that the polypeptide a chain in the multispecific antibody of Figure 1 is replaced by a polypeptide a-33 chain. The polypeptide a-33 chain is designed so that the variable region Va1 binds to a single-chain antibody ScFv1 and the constant region Ca2 binds to a single-chain antibody ScFv2 having a different CDR from that of the single-chain antibody ScFv1, and so that the CDR of the Fv consisting of the variable region Va2 and the variable region Vb is shared with the CDR of the Fv consisting of the variable region Va1 and the variable region Vb but is different from the CDRs of both the single-chain antibody ScFv1 and the single-chain antibody ScFv2.

[0062] In multispecific antibodies 31, 32, and 33 in Figures 11 to 13, each Fv specific for a particular epitope is enclosed by a dashed line. For example, in multispecific antibodies 31 and 32, of the three Fvs in total, two Fvs in both arms of the antibody are shared, and an Fv corresponding to an epitope different from these Fvs is carried by single-chain antibody ScFv1 or single-chain antibody ScFv2. In other words, multispecific antibodies 31 and 32 are bispecific antibodies, but are designed to have an enhanced binding valency for one of the target epitopes. In addition, in multispecific antibody 33, of the four Fvs in total, two Fvs in both arms of the antibody are shared, and an Fv corresponding to two epitopes different from these Fvs is carried by single-chain antibody ScFv1 and single-chain antibody ScFv2, respectively. In other words, multispecific antibody 33 is a trispecific antibody, but is designed to have an enhanced binding valency for one of the target epitopes. In more specific examples of these multispecific antibodies 31, 32, and 33, the two Fvs in each arm of the antibody can be configured to be specific to cancer cells, and the single-chain antibody ScFv1 and / or single-chain antibody ScFv2 can be configured to be specific to immune cells. Such antibodies can be used to suppress cancer cell-independent activation of immune cells and enhance cytotoxicity against cancer cells.

[0063] In the polypeptide a-31 chain of multispecific antibody 31, either the VH or VL of single-chain antibody ScFv1 may be linked to the variable region Va1. Similarly, in the polypeptide a-32 chain of multispecific antibody 32, either the VH or VL of single-chain antibody ScFv2 may be linked to the constant region Ca2. Similarly, in the polypeptide a-43 chain of multispecific antibody 43, either the VH or VL of single-chain antibody ScFv1 may be linked to the variable region Va1, and either the VH or VL of the independent single-chain antibody ScFv2 may be linked to the constant region Ca2.

[0064] The sequence and length of the peptide linker linking VH and VL constituting the single-chain antibody ScFv1, 2; the linking mode between the single-chain antibody ScFv1, 2 and the variable region Va1 or the constant region Ca2; and the sequence and length of the peptide linker linking the single-chain antibody ScFv1, 2 and the variable region Va1 or the constant region Ca2 are as described above in "1-6. Multispecific antibodies 21, 22, 23."

[0065] The specific sequences of the variable region Va1, constant region Ca1, variable region Va2, and constant region Ca2 in the polypeptide a-31 chain, polypeptide a-32 chain, and polypeptide a-33 chain constituting each of the multispecific antibodies 31 to 33, and the variable region Vb and constant region Cb in the polypeptide b chain, may each independently be the sequences of either the heavy chain or the light chain.

[0066] Therefore, in one example of multispecific antibodies 31 to 33, the variable region Va1, constant region Ca1, variable region Va2, and constant region Ca2 in the polypeptide a-31 chain, polypeptide a-32 chain, and polypeptide a-33 chain may all have heavy chain sequences, and the variable region Vb and constant region Cb of the polypeptide b chain may all have light chain sequences, similar to multispecific antibody 12 in Figure 5. Furthermore, in another example of multispecific antibodies 31 to 33, the variable region Va1 and variable region Va2 in the polypeptide a-31 chain, polypeptide a-32 chain, and polypeptide a-33 chain may all have heavy chain sequences, and the constant regions Ca1 and Ca2 have light chain sequences, similar to multispecific antibody 13 in Figure 7. In addition, in the polypeptide b chain, the variable region Vb may have a light chain sequence and the constant region Cb may have a heavy chain sequence.

[0067] As further variations of the multispecific antibodies 31 to 33, the peptide linker LL may be replaced with a peptide linker LL-11 containing a protease recognition sequence, or the Fc region may be absent.

[0068] 1-8. Multispecific antibody 11' The multispecific antibodies of the present invention may be those in which the peptide linker LL, which contributes to the formation of their characteristic formats, has been cleaved. The manner in which the peptide linker LL is cleaved is not particularly limited, but a preferred embodiment is one in which a protease sequence introduced into the peptide linker LL is specifically cleaved by the corresponding protease.

[0069] Examples of multispecific antibodies of the present invention in this cleavage mode include multispecific antibodies having a Fab region comprising one predetermined polypeptide a' chain, one predetermined polypeptide a" chain, and two predetermined polypeptide b chains. The predetermined polypeptide a' chain comprises a polypeptide in which a variable region Va1, a constant region Ca1, and a cleavage fragment Lr1' of the protease recognition sequence Lr1 are linked in this order. The predetermined polypeptide a" chain comprises a polypeptide in which a cleavage fragment Lr2' of the protease recognition sequence Lr2, a variable region Va2, and a constant region Ca2 are linked in this order. The predetermined polypeptide b chain comprises a polypeptide in which a variable region Vb is linked to a constant region Cb that binds to the constant region Ca1 or the constant region Ca2.

[0070] As a specific example of such a multispecific antibody of the present invention, an example of an IgD,E,G bispecific antibody (multispecific antibody 11') comprising a cleavable peptide linker is schematically shown in Figure 14. Multispecific antibody 11' has a structure in which the protease recognition sequences Lr1 and Lr2 introduced into the peptide linker LL in multispecific antibody 11 of Figure 3 are specifically cleaved by the corresponding protease, splitting the polypeptide a chain into polypeptide a' chain and polypeptide a'' chain, leaving behind a cleavage fragment Lr1' derived from the protease recognition sequence Lr1 and a cleavage fragment Lr2' derived from the protease recognition sequence Lr2.

[0071] As described for multispecific antibodies 21 to 23 in Figures 9 to 11, the specific sequences of the variable region Va1, constant region Ca1, variable region Va2, and constant region Ca2 of the polypeptide a' chain and polypeptide a'' chain constituting multispecific antibody 11', and the variable region Vb and constant region Cb of the polypeptide b chain, may each independently be the sequences of either the heavy chain or the light chain.

[0072] As a further modification of the multispecific antibody 11', the Fc region may be absent.

[0073] 2. DNA The DNA of the present invention encodes a polypeptide a chain comprising a polypeptide in which a variable region Va1, a constant region Ca1, a peptide linker LL, a variable region Va2, and a constant region Ca2 are linked in this order. The DNA of the present invention is used to produce the above-mentioned "1. Multispecific antibodies." Therefore, the DNA of the present invention is used together with DNA encoding a polypeptide b chain comprising a polypeptide in which a variable region Vb is linked to a constant region Cb that binds to the constant region Ca1 or the constant region Ca2.

[0074] Polypeptide a chain and polypeptide b chain are as described in detail above in "1. Multispecific antibodies." Specific embodiments of the polypeptide a chain, such as the polypeptide a-11 chain (an embodiment comprising a protease recognition sequence), polypeptide a-12 chain (an embodiment in which the heavy chain / light chain origin of each region is specified), polypeptide a-13 chain (an embodiment in which the heavy chain / light chain origin of each region is specified), polypeptide a-21 chain (an embodiment comprising a single-chain antibody), polypeptide a-22 chain (an embodiment comprising a single-chain antibody), and polypeptide a-23 chain (an embodiment in which a single-chain antibody) are also as described in detail above in "1. Multispecific antibodies."

[0075] Those skilled in the art can appropriately design the base sequences of the DNA encoding the polypeptide a chain and the DNA encoding the polypeptide b chain based on the design of the polypeptide a chain and the polypeptide b chain. Furthermore, the DNA encoding the polypeptide a chain and the DNA encoding the polypeptide b chain can be obtained by artificial synthesis using genetic engineering techniques.

[0076] These DNAs are preferably those whose codon usage frequency is optimized for the host. For example, when human cells are used as the host, DNAs whose codon usage frequency is optimized for human cells are preferred.

[0077] 3. Recombinant Vector The DNA encoding the polypeptide a chain and the DNA encoding the polypeptide b chain described in "2. DNA" above can each be incorporated into an expression vector. An expression vector incorporating DNA encoding the polypeptide a chain is referred to as recombinant vector va, and an expression vector incorporating DNA encoding the polypeptide b chain is referred to as recombinant vector vb.

[0078] Schematic diagrams of the recombinant vectors va and vb are shown in Figure 15. The recombinant vectors va and vb shown in Figure 15 are examples of those used to produce multispecific antibody 1 in Figure 1. In Figure 15, "S" represents the signal sequence, "Va1" represents the DNA encoding the variable region Va1, "Ca1" represents the DNA encoding the constant region Ca1, "LL" represents the DNA encoding the peptide linker LL, "Va2" represents the DNA encoding the variable region Va2, "Ca2" represents the DNA encoding the constant region Ca2, "Vb" represents the DNA encoding the variable region Vb, "Cb" represents the DNA encoding the constant region Cb, "H" represents the DNA encoding the hinge region, "CH2" represents the DNA encoding the constant region CH2, and "CH3" represents the DNA encoding the constant region CH3.

[0079] The recombinant vector v a contains a regulatory element such as a promoter operably linked to the DNA encoding the polypeptide a chain. Similarly, the recombinant vector v b contains a regulatory element such as a promoter operably linked to the DNA encoding the polypeptide b chain. A typical regulatory element is a promoter, but it may also contain transcription elements such as an enhancer, CCAAT box, TATA box, or SPI site, as necessary. Furthermore, "operably linked" means that the DNA of the present invention is linked to various regulatory elements such as promoters and enhancers that regulate the DNA encoding the polypeptide a chain or polypeptide b chain in a state that allows them to operate in a host cell.

[0080] Suitable expression vectors are those constructed for genetic recombination from phages, plasmids, or viruses that can autonomously replicate in a host. Such expression vectors are well known, and examples include pUC vectors, pBluescript vectors, pET vectors, pGEX vectors, pEX vectors, and pCAGGS vectors. An expression vector may be used in combination with an appropriate host cell.

[0081] 4. Transformants The transformant of the present invention can be obtained by transforming a host using the DNA encoding the polypeptide a chain and the DNA encoding the polypeptide b chain described in "2. DNA" above, or the recombinant vector va and recombinant vector vb described in "3. Recombinant Vector" above.

[0082] The host used to produce the transformant can be any prokaryotic or eukaryotic cell, as long as it can be introduced with a gene, is capable of autonomous replication, and is capable of expressing the multispecific antibody of the present invention. Specific host cells include mammalian cells such as CHO cells, N50 cells, SP2 / 0 cells, Expi293 cells, HEK293 cells, COS cells, and PER.C6 cells; fungi such as yeast; and bacteria such as Escherichia coli (E. coli).

[0083] The transformant of the present invention can be produced by introducing the above-mentioned DNA or recombinant vector into a host. The method for introducing these nucleic acid species is not particularly limited as long as the gene of interest can be introduced into the host. The location where the above-mentioned DNA is introduced is also not particularly limited as long as the gene of interest can be expressed, and may be on a plasmid or on the genome. Specific methods for introducing the above-mentioned DNA or recombinant vector include, for example, recombinant vector methods and genome editing methods.

[0084] The conditions for introducing the DNA or recombinant vector into the host may be appropriately set depending on the introduction method, the type of host, etc. When the host is an animal cell, examples of the method include the polyethyleneimine method, electroporation, calcium phosphate method, and lipofection method. When the host is a fungus, examples of the method include the electroporation method, spheroplast method, and lithium acetate method. When the host is a bacterium, examples of the method include a method using competent cells treated with calcium ions and the electroporation method.

[0085] The ratio of the DNA or recombinant vector introduced into the host may be, for example, 0.4 or 0.5 moles or more of the DNA or recombinant vector va encoding the polypeptide a chain per mole of the DNA or recombinant vector vb encoding the polypeptide b chain, preferably 0.7 moles or more, more preferably 0.9 moles or more or 1 mole or more, and even more preferably 1.2 moles or more, 1.25 moles or more, 1.35 moles or more, 1.4 moles or more, or 1.5 moles or more. The upper limit of the molar ratio of the recombinant vector va to 1 mole of the recombinant vector vb is not particularly limited, but may be, for example, 3 moles or less, 2.5 moles or less, 2 moles or less, or 1.8 moles or less. Using these amounts is preferable in that it reduces the amount of tetramer 12BQ of the polypeptide b-12 chain, as shown in Figure 6, produced as a by-product.

[0086] 5. Method for producing multispecific antibodies The method for producing a multispecific antibody of the present invention comprises an antibody production step of culturing the transformant of the present invention.

[0087] The culture conditions for the antibody production step may be appropriately determined taking into consideration the nutritional and physiological properties of the host, but liquid culture is preferred. Furthermore, from the perspective of industrial production, culture is preferably carried out under aeration and agitation conditions.

[0088] In the antibody production process, the polypeptide a chain and the polypeptide b chain are expressed and naturally associate to form the multispecific antibody of the present invention. For example, in the antibody production process in the method for producing multispecific antibody 1 shown in Figure 1, the polypeptide a chain and the polypeptide b chain shown in Figure 2 are expressed and naturally associate to form multispecific antibody 1. Depending on the specific sequences of the polypeptide a chain and the polypeptide b chain, by-products may be generated as shown in Figure 6, but these by-products do not exhibit immunoreactivity.

[0089] When the multispecific antibody produced in the antibody production step contains a protease recognition sequence in the peptide linker LL, the multispecific antibody can be subjected to a linker cleavage step in which the peptide linker LL is cleaved using a protease corresponding to the protease.

[0090] For example, when the peptide linker LL in the multispecific antibody produced in the antibody production step contains a protease recognition sequence Lr1 on the constant region Ca1 side and a protease recognition sequence Lr2 on the variable region Va2 side as shown in FIG. 3, the peptide linker LL is cleaved in the linker cleavage step using proteases corresponding to the protease recognition sequence Lr1 and the protease recognition sequence Lr2, respectively.

[0091] Furthermore, when protease recognition sequences Lr1 and Lr2 are designed so that cleavage by the corresponding proteases leaves cleavage fragments Lr1' and Lr2', respectively, on the multispecific antibody side, cleavage of the peptide linker LL yields multispecific antibody 11' as shown in Figure 14. When protease recognition sequences Lr1 and Lr2 are designed so that cleavage by the corresponding proteases does not leave cleavage fragments Lr1' and / or Lr2', respectively, on the multispecific antibody side, cleavage of the peptide linker LL yields multispecific antibody 11'' (although not shown, an embodiment in which Lr1' is absent and Lr2' remains) or multispecific antibody 11''' as shown in Figure 16.

[0092] The multispecific antibodies of the present invention obtained in the antibody production step or the linker-cleaved antibody step can be further purified by subjecting them to a purification step. The purification step can be performed using known antibody purification methods, such as centrifugation, affinity chromatography (protein A affinity chromatography, protein G affinity chromatography, etc.), size exclusion chromatography, ion exchange chromatography (cation exchange chromatography, anion exchange chromatography), hydrophobic interaction chromatography, gel electrophoresis, and dialysis. Preferably, protein A affinity chromatography is used in combination with ion exchange chromatography (preferably cation exchange chromatography).

[0093] Because the multispecific antibodies of the present invention are designed to be free of immunoreactive by-products, the purification process does not require the specialized and complicated separation steps typically used in the purification of multispecific antibodies. In other words, the method for producing multispecific antibodies of the present invention can be simplified, making it extremely advantageous for industrial production.

[0094] 6. Multispecific antibody production kit The present invention also provides a multispecific antibody production kit for producing the above-mentioned "1. Multispecific antibodies." The multispecific antibody production kit of the present invention can be used to produce the above-mentioned "3. Recombinant vector" or "4. Transformant," or to carry out the above-mentioned "5. Method for producing multispecific antibodies."

[0095] The multispecific antibody production kit of the present invention comprises: an expression vector va' comprising, in this order: a cloning site CS1 for incorporating the variable region Va1, DNA encoding the constant region Ca1, DNA encoding the peptide linker LL, a cloning site CS2 for incorporating the variable region Va2, and DNA encoding the constant region Ca2; and an expression vector vb' comprising a cloning site CS for incorporating the variable region Vb, and DNA encoding the constant region Cb that binds to the constant region Ca1 or the constant region Ca2.

[0096] Figure 17 shows a schematic diagram of the expression vectors included in the multispecific antibody production kit of the present invention. In the expression vector va', the restriction enzyme sites contained in the cloning site CS1 and the cloning site CS2 are designed to be specific for different restriction sequences so that the variable regions Va1 and Va2 can be incorporated, respectively. Each of the cloning sites CS1, CS2, and CS may contain one restriction enzyme site, or may be a multicloning site containing two or more restriction enzyme sites. The restriction enzyme sites contained in these cloning sites are appropriately selected from known restriction enzyme sites. Furthermore, the multicloning site may be a multicloning site contained in a known cloning vector or expression vector, or may be an appropriately modified known multicloning site.

[0097] The method for incorporating the variable regions Va1, Va2, and Vb into the cloning sites CS1, CS2, and CS can be appropriately determined by those skilled in the art based on known cloning methods using restriction enzymes appropriate for the cloning sites CS1, CS2, and CS.

[0098] 7. Diagnostic and Pharmaceutical Compositions The multispecific antibodies of the present invention can be used in any application that utilizes their specific binding ability to two or more different epitopes. Examples of applications that utilize the specific binding ability of the multispecific antibodies of the present invention include targeting multiple antigens (cytokines, tumor markers), targeting different epitopes on the same tumor or the same viral antigen, and cross-linking two target cells (e.g., bringing immune effector cells into close proximity with specific tumor-associated antigens to promote cell death). Therefore, the multispecific antibodies of the present invention are useful for diagnostic applications and pharmaceutical applications (so-called antibody drugs).

[0099] Therefore, the multispecific antibodies of the present invention can be used as an active ingredient in a diagnostic agent (sensing component) or pharmaceutical composition. That is, the present invention provides a diagnostic agent comprising the multispecific antibody described above in "1. Multispecific antibodies," and a pharmaceutical composition comprising the multispecific antibody described above in "1. Multispecific antibodies."

[0100] Examples of diagnostic agents of the present invention include compositions in which a multispecific antibody is combined with other components commonly used in diagnostic agent compositions (e.g., buffers, suspending agents, stabilizers, preservatives, antiseptics, etc.), and compositions in which a multispecific antibody is immobilized on the surface of an insoluble carrier (particle or substrate).

[0101] Examples of pharmaceutical compositions of the present invention include compositions in which a multispecific antibody is composed together with other ingredients commonly used in pharmaceutical compositions (e.g., excipients, buffers, suspending agents, stabilizers, preservatives, antiseptics, physiological saline, etc.). [Example]

[0102] The present invention will be described in detail below with reference to examples, but the present invention should not be construed as being limited to these examples. Note that hereinafter, the multispecific antibodies of the present invention, that is, multispecific monoclonal antibodies having a Fab region comprising one polypeptide a chain (comprising a polypeptide in which a variable region Va1, a constant region Ca1, a peptide linker LL, a variable region Va2, and a constant region Ca2 are linked in this order) and two polypeptide b chains (comprising a polypeptide in which a variable region Vb is linked to a constant region Cb that binds to the constant region Ca1 or the constant region Ca2), will also be referred to as TribsMab CLC (Trimeric Bi-specific Monoclonal Antibody Common Light Chain).

[0103] Test Example 1: Design and production of multispecific antibody (anti-HER2 x HER3 bispecific antibody-1) (1) Design of anti-HER2×HER3 bispecific antibody (HER2×HER3 TribsMab CLC) A bispecific antibody corresponding to multispecific antibody 12 in Figure 5 was designed. In this test example, the bispecific antibody was designed to target HER2 and HER3 expressed on the surface of tumor cells. The variable region (the area enclosed by the dashed line in Figure 5) had the same sequence as the variable region of MCLA-128, a known anti-HER2 x Her3 bispecific antibody (Cancer Cell 33, 922-936 (2018)), i.e., the heavy chain variable region 3958VH (specific for HER2) and the heavy chain variable region 3178VH (specific for HER3) of MCLA-128, and the light chain variable region 128VL of MCLA-128. Furthermore, a sequence derived from the human IgG1 class was used for the constant region. Furthermore, peptide linkers of different lengths were designed to correspond to the peptide linker LL in Figure 5, with the basic sequence GGGGS and with or without the HRV3C protease recognition sequence (LEVLFQGP).

[0104] The correspondence between each domain of multispecific antibody 12 in Figure 5 and the bispecific antibody designed in this test example is shown in Tables 1 and 2 below, and a schematic diagram of the bispecific antibody designed in this test example is shown in Figure 18. The specific lengths (Å) of peptide linkers of different lengths and their specific sequences are shown in Table 1. Each peptide linker is represented as L(x), where "x" in parentheses indicates the total number of amino acid residues constituting the peptide linker if it contains a protease recognition sequence (for example, a peptide linker that contains a protease recognition sequence and has a total of 68 amino acid residues constituting the peptide linker is represented as "L(68)"), and delP is added if it does not contain a protease recognition sequence.

[0105] [Table 1] [Table 2]

[0106] (2) Construction of recombinant vectors for expressing bispecific antibodies Figure 19 shows a schematic diagram of the recombinant vector va for the a-12 chain and the recombinant vector vb for the b-12 chain, used to express the bispecific antibody designed in (1) above. In the diagram, S represents the signal sequence, and H represents the hinge region. The expression vector used to incorporate the DNA encoding each chain was pCAGGS, an expression vector for mammalian cells that contains the chicken β-actin gene promoter and the cytomegalovirus CMV-IE enhancer (CAG promoter). Furthermore, a leader sequence for secretory expression of the antibody was incorporated into the N-terminus of the DNA encoding each chain. The sequences of the resulting recombinant vectors va and vb were confirmed by sequencing.

[0107] (3) Preparation of transformants Human embryonic kidney cells, Expi293F cells, were used as hosts and transfected using the polyethyleneimine method. All cells were cultured at 37°C, 5% CO2, and 125 rpm.

[0108] 6 μg of expression vector va and 12 μg of expression vector vb (the expression vector introduction ratio va:vb is approximately 0.5:1 on a molar basis) were added to 900 μL of Opti-MEM. (R) (Thermo) and Opti-MEM (R) A solution was prepared by adding 75 μL of PEI-MAX (Polysciences) to the 100 μL PBS solution. These two solutions were mixed and left to stand for 20 minutes, then added to 16.2 mL of HE400 (Gmep) to prepare a transfection solution. Host cells were cultured in an Erlenmeyer flask containing 30 mL of HE200 medium (Gmep) until the cell count reached 3-5 × 10 6 cells / mL, and when the viability reached 95% or higher, the cell count was 45 × 10 6 The cell culture medium was dispensed into 50 mL sample tubes so that the cells were 1000 cells. After centrifuging the sample tubes at 1,500 rpm for 5 minutes, the culture medium was removed and the cells were suspended in transfection solution. 3 mL of the cell suspension was seeded into a 6-well plate and cultured for 20 hours.

[0109] (4) Production of bispecific antibodies 7.5 μL of 0.5 M sodium valproate solution and 12 μL of 1 M sodium propionate solution were added to each well, and the cells were cultured for an additional 6 days under the culture conditions described in (3) above. The culture supernatant was subjected to polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using an anti-human Fc antibody to confirm the expression of the bispecific antibody.

[0110] (5) Purification of bispecific antibodies (5-1) Protein A affinity purification The culture medium was centrifuged at 6,000 rpm at 4°C for 10 minutes to remove the cells, and the culture supernatant was filtered through a 0.45 μm pore size filter (Millex-HP, Millipore). TM The culture supernatant filtrate was added to the column (column volume: 500 μL), washed with TBS, and eluted with Gentle Ag / Ab Elution Buffer (Thermo).

[0111] Each eluted fraction was subjected to SDS-PAGE under reducing and non-reducing conditions and then subjected to CBB staining. The results under reducing conditions are shown in Figure 20, and the results under non-reducing conditions are shown in Figure 21.

[0112] The molecular weights were calculated from the electrophoretic mobility of the bands observed in Figure 20 (reducing conditions) and compared with the molecular weights based on the amino acid sequences of the polypeptide a-12 chain and polypeptide b-12 chain. The results are shown in Table 3. The results in Table 3 confirmed the identity of the polypeptide a-12 chain and polypeptide b-12 chain that constitute the bispecific antibody of interest (multispecific antibody 12).

[0113] [Table 3]

[0114] Furthermore, the molecular weight was calculated based on Figure 21 (non-reducing conditions), and it was found that in addition to the desired bispecific antibody (multispecific antibody 12), a dimer of the polypeptide b-12 chain, 12BD, was present as a major by-product.

[0115] (5-2) Analysis of the product by gel filtration chromatography The fraction containing the bispecific antibody of interest (multispecific antibody 12) obtained in Section 5-1 above was loaded onto a gel filtration chromatography column, Superdex 200 10 / 300 GL (GE Healthcare), equilibrated with PBS. Elution was performed using an AKTA Prime plus (GE Healthcare) at a flow rate of 0.5 mL / min. Ferritin, conalbumin, aldolase, and ovalbumin were used as molecular weight markers.

[0116] The results of gel filtration chromatography for Example 1 are shown in Figure 22. The eluted fractions from 16 mL to 18.5 mL were subjected to SDS-PAGE (non-reducing conditions) in the same manner as in 5-1 above, and the results are shown in Figure 23.

[0117] The gel filtration chromatogram in Figure 22 shows a peak for the bispecific antibody (multispecific antibody 12) and a peak believed to be tetrameric 12BQ of the polypeptide b-12 chain. This inference is based on the fact that the peak for multispecific antibody 12 in Figure 22 corresponds to the band in Figure 23 where multispecific antibody 12 is observed, and that the peak believed to be tetrameric 12BQ in Figure 22 corresponds to the band in Figure 23 where dimeric BD (detected in a dissociated state of tetrameric 12BQ due to the SDS-PAGE conditions) is observed. In other words, it was found that, along with the desired multispecific antibody 12, something believed to be tetrameric 12BQ was produced as a by-product.

[0118] Furthermore, the areas of each peak in Figure 22 were calculated using PrimeView Evaluation (GE Healthcare), and the results showed that the peak area proportion of multispecific antibody 12 was approximately 67%, and that of tetramer 12BQ was approximately 32%.

[0119] Furthermore, the results of similar gel filtration chromatography for multispecific antibody 12 of Examples 2 to 4 are shown in Figure 24, and the results of similar gel filtration chromatography for multispecific antibody 12 of Example 5 are shown in Figure 25.

[0120] 22 and 24, the proportion of by-products produced was lowest in the production of multispecific antibody 12 in Example 1, which had the longest peptide linker having a protease recognition sequence, and the proportion of by-products produced tended to increase as the length of the peptide linker having a protease recognition sequence became shorter (Examples 2 to 4). In other words, when a peptide linker having a protease recognition sequence is used, the amount of by-products produced tends to decrease as the length of the peptide linker becomes longer.

[0121] On the other hand, as shown in FIG. 25, when the peptide linker did not have a protease recognition sequence (Example 5), no by-products were observed to be produced.

[0122] (5-3) Affinity purification using IgG-CH1-binding carrier CaptureSelect equilibrated with PBS TM The fraction containing the bispecific antibody of interest (multispecific antibody 12) obtained in 5-1 above was applied to IgG-CH1 Affinity Matrix (Thermo) (column volume: 250 μL), washed with PBS, and then eluted with acetate buffer (20 mM CH3COOH, 150 mM NaCl, pH 3.5). Each eluted fraction during the purification process was subjected to SDS-PAGE (non-reducing conditions) as in 5-1 above. The results are shown in Figure 26. In Figure 26, lane 1 represents the data after Protein A affinity purification but before generation of the IgG-CH1-binding carrier; lane 2 represents the flow-through fraction; lane 3 represents the wash fraction; lane 4 represents elution fraction 1; and lane 5 represents elution fraction 2.

[0123] As is clear from Figure 26, the by-product tetrameric BQ was eluted in the flow-through fraction, and therefore these by-products could be separated from the target multispecific antibody 12.

[0124] (5-4) Affinity purification using cation exchange chromatography Using AKTA Prime plus, the fraction containing the bispecific antibody of interest (multispecific antibody 12) obtained in 5-1 above was applied to a cation exchange column, Resource S (GE Healthcare), and elution was performed using a gradient of 10 mM MES buffer (pH 6.0) as the running buffer and 10 mM MES buffer (pH 6.0) containing 1 M NaCl as the elution buffer, with a final elution buffer concentration of 40%. The results are shown in Figure 27.

[0125] As is clear from Figure 27, the by-product tetrameric BQ could be clearly separated from the target multispecific antibody 12.

[0126] Test Example 2: Evaluation of activity of multispecific antibody (anti-HER2 × HER3 bispecific antibody-1) (1) Binding activity evaluation-1 Multispecific antibodies 12 of Examples 1 to 5 prepared in Test Example 1 were evaluated for their binding activity against HER2- and HER3-positive human breast adenocarcinoma cells MCF-7 by flow cytometry as follows.

[0127] MCF-7 cells were detached from the dish using trypsin / EDTA (0.25 w / v% trypsin-1 mmol / L EDTA 4Na solution, containing phenol red, Gibco), and then collected by centrifugation at room temperature at 1,000 rpm for 5 minutes. 5 × 10 cells were collected. 5 The cells were dispensed into sample tubes. After washing once with 0.1% NaN3 / PBS, multispecific antibody 12, 3958 antibody (anti-HER2 antibody), and 3178 antibody (anti-HER3 antibody) (500 nM) were added as primary antibodies and incubated on ice for 30 minutes. After washing once, 1 μL of 2 mg / mL anti-human IgG (Fc-specific)-FITC antibody (Sigma Aldrich) solution was added as secondary antibody and 499 μL of 0.1% NaN3 / PBS was added and incubated on ice for 30 minutes. After washing once, the cells were suspended in 500 μL of 0.1% NaN3 / PBS, filtered through a nylon filter, and dispensed into plastic tubes. BD Accuri TMFlow cytometry analysis was performed using C6 (BD Biosciences). As a result, it was confirmed that all of the antibodies of Examples 1 to 5 exhibited binding activity to HER2- and HER3-positive human breast adenocarcinoma cells MCF-7.

[0128] (2) Binding activity evaluation-2 Multispecific antibodies 12 of Examples 1 to 4, which had different peptide linker lengths, were subjected to binding activity assessment against MCF-7 cells by flow cytometry in the same manner as in (1) above, except that their concentrations were changed from 500 nM to 1 nM, 10 nM, 100 nM, or 1000 nM. The results are shown in Figure 28.

[0129] As shown in Figure 28, a difference in binding activity was observed when the antibody concentration was 10 nM. Specifically, the fluorescence intensities of Example 4 (L(23)) and Example 3 (L(38)) were almost equivalent, but the fluorescence intensities were 1.5 times that of Example 2 (L(53)) and twice that of Example 1 (L(68)).

[0130] The difference in binding activity due to differences in peptide linker length is thought to be due to differences in binding mode. HER2 and HER3 present on the surface of MCF-7 exist either singly or as heterodimers. On the surface of cells where HER2 and HER3 expression levels are considered to be equal, such as MCF-7, it is thought that HER2 and HER3 are present as heterodimers at a particularly high rate. Furthermore, the shorter the peptide linker length, the more restricted the range of motion of the Fab arm of the multispecific antibody, which facilitates bivalent binding to the HER2 and HER3 heterodimer, presumably resulting in enhanced cell binding compared to monovalent binding. It is thought that the influence of bivalent binding was more pronounced under conditions of low antibody concentration.

[0131] (3) Bispecificity evaluation The bispecificity of multispecific antibody 12 of Example 1 was evaluated using surface plasmon resonance (SPR) as follows.

[0132] The Fc-fused HER2 extracellular domain (ECD) was immobilized on the surface of a Biacore sensor chip CM5 using an amine coupling kit (GE Healthcare). Multispecific antibody 12 (1 μM) from Example 1 and Fc-fused HER3 ECD (1 μM) were added to the Fc-fused HER2 ECD-immobilized sensor chip in this order at a flow rate of 20 μL / min at 25°C, and SPR analysis was performed using a Biacore3000 (GE Healthcare). PBS containing 0.005% Tween-20 (PBST) was used as the running buffer. The results are shown in Figure 29.

[0133] 29, when the bispecific antibody of Example 1 (multispecific antibody 12) was added to the Fc-fused HER2ECD-immobilized sensor chip, the response, indicating an increase in mass on the sensor chip, increased, and when Fc-fused HER3ECD was added, the response also increased. In other words, it was confirmed that the bispecific antibody of Example 1 (multispecific antibody 12) has bispecificity for the antigens HER2 and HER3.

[0134] (4) Cell proliferation inhibitory activity MCF-7 cells were cultured in D-MEM (Dulbecco's Modified Eagle Medium, Sigma-Aldrich) containing 10% FBS. Cells were harvested from the dish using trypsin / EDTA (0.25 w / v% trypsin-1 mmol / L EDTA 4Na solution, containing phenol red, Gibco) and cultured at 6.25 × 10 cells / well in RPMI 1640 (Roswell Park Memorial Institute medium, Sigma-Aldrich) containing 1% FBS. 3The cells were suspended at a concentration of 1000 cells / mL and 80 μL of each was seeded into wells of a 96-well flat-bottom culture plate. 10 μL of recombinant human heregulin β-1 (carrier-free) (TONBO biosciences) diluted to 0.1 ng / μL with PBS was added to each well, and 10 μL of each of multispecific antibody 12, antibody 3958 (anti-HER2 antibody), and antibody 3178 (anti-HER3 antibody) prepared at concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM, and 1000 nM, respectively, of Examples 1 to 5 was added to each well. After 6 days of incubation at 37°C in 5% CO2, viable cells were quantified colorimetrically using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay with CellTiter 96 AQueous One Solution Reagent (Promega). Absorbance at 490 nm was measured using an iMark Microplate Reader (BioRad). The background absorbance at 630 nm was subtracted, and the cell proliferation rate relative to MCF-7 cells was calculated using the following formula (A490 target maximum control refers to the absorbance of MCF-7 cells without antibody). The results are shown in Figure 30.

[0135]

number

[0136] As shown in Figure 30, all of the bispecific antibodies (multispecific antibody 12) of Examples 1 to 5 exhibited the ability to inhibit cell proliferation against MCF-7, and it was found that the cell proliferation rate decreased with increasing antibody concentration. In other words, it was shown that all of the bispecific antibodies (multispecific antibody 12) of Examples 1 to 5 inhibited the proliferation signaling of MCF-7. Furthermore, since no significant difference in cell proliferation inhibitory ability was observed among Examples 1 to 5, it is thought that there is no significant difference in proliferation inhibitory ability due to differences in linker length.

[0137] Test Example 3: Investigation of production conditions for multispecific antibodies Multispecific antibodies were prepared in the same manner as in Example 1, except that the introduction ratios of the recombinant vectors va and vb prepared in Test Example 1 were changed, and the amount of tetramer 12BQ produced was confirmed by gel filtration chromatogram in the same manner as in Test Example 1 (5-2). The introduction ratios (by weight) of the recombinant vectors va and vb used in this Test Example are shown in the table below. Note that the introduction ratios (by weight) of the recombinant vectors va and vb shown in the table below are almost the same as the introduction ratios on a molar basis. The table also lists the introduction ratios in Example 1 prepared in Test Example 1.

[0138] [Table 4]

[0139] As a result, the amount of tetrameric 12BQ produced decreased in the order of Example 1, Example 6, and Example 7, and was approximately the same in Example 7 and Example 8. In other words, it was found that the amount of tetrameric 12BQ produced as a by-product tends to decrease as the amount of recombinant vector va introduced increases relative to the theoretical amounts of recombinant vector va and recombinant vector vb introduced (Example 1).

[0140] Test Example 4: Design and production of multispecific antibody (anti-HER2×HER3 bispecific antibody-2) (1) Design of HER2×HER3 bispecific antibodies A bispecific antibody corresponding to multispecific antibody 13 in Figure 7 was designed. Specifically, multispecific antibody 13 (Example 9) was designed in the same manner as Example 1 in Test Example 1, except that the heavy chain constant region CHa1 and the light chain constant region CHb of multispecific antibody 12 prepared in Example 1 of Test Example 1 were swapped, and the heavy chain constant region CHa2 and the light chain constant region CHb were swapped. The correspondence between the domains of multispecific antibody 13 (Example 9) in Figure 7 and the bispecific antibody designed in this Test Example is shown in Tables 5 and 6 below, and a schematic diagram of the bispecific antibody designed in this Test Example (Example 9) is shown in Figure 31.

[0141] [Table 5]

[0142] [Table 6]

[0143] (2) Construction of recombinant vectors for expressing bispecific antibodies Schematic diagrams of the recombinant vector va for the a-13 chain and the recombinant vector vb for the b-13 chain, used to express the bispecific antibody designed in (1) above, are shown in Figure 32. These recombinant vectors are also similar to the recombinant vectors prepared in Example 1 in Test Example 1, except that they were designed by swapping the region encoding the heavy chain constant region CHa1 and the region encoding the light chain constant region CHb of multispecific antibody 12 in the recombinant vector for expressing the bispecific antibody of Example 1 in Test Example 1, and swapping the region encoding the heavy chain constant region CHa2 and the region encoding the light chain constant region CHb.

[0144] (3) Creation, production, and purification of transformants Transformants were prepared, produced, and purified (protein A affinity purification) in the same manner as in Test Example 1. The purified fraction was subjected to SDS-PAGE under reducing and non-reducing conditions in the same manner as in Test Example 1, and the results are shown in Figure 33.

[0145] As shown in a comparison between Figure 33 (Example 9) and Figure 21 (Example 1), in Example 9, no dimer (dissociated tetramer) by-product was observed, and almost only the desired bispecific antibody (multispecific antibody 13) was confirmed.

[0146] Furthermore, the purified fraction was subjected to gel filtration chromatography analysis in the same manner as in Test Example 1, and the results are shown in Figure 34. Figure 34 shows the results of Example 9 together with the results of Example 1. As shown in Figure 34, the tetramer by-product (peak indicated by an arrow in the figure) that was confirmed in Example 1 was not detected in Example 9.

[0147] Test Example 5: Design and production of multispecific antibodies (anti-CD20 x CD3 bispecific antibody, anti-BCMA x CD3 bispecific antibody) (1) Design of bispecific antibodies (1-1) Design of anti-CD20×CD3 bispecific antibody (CD20×CD3 TribsMab CLC) A bispecific antibody corresponding to multispecific antibody 12 in Figure 5 was designed. The bispecific antibody was designed to target CD20 and CD3. The variable region (the area enclosed by the dashed line in Figure 5) had the same sequence as the variable region of REGN1979 (Eric J. Smith, Kara Olson, Lauric J. Haber, Bindu Varghese, Paurene Duramad. Sci Rep, 5, 17943 (2016)), a known anti-CD20 x CD3 bispecific antibody. That is, the heavy chain variable region 1979VH-CD20 (specific for CD20) and the heavy chain variable region 1979VH-CD3 (specific for CD3) of REGN1979, as well as the light chain variable region 1979VL of REGN1979 were used. The light chain class of REGN1979 is λ. The constant region used was derived from the human IgG1 class. Furthermore, a peptide linker with GGGGS as the basic sequence was designed as the sequence corresponding to the peptide linker LL in FIG.

[0148] The correspondence between each domain of multispecific antibody 12 in Figure 5 and the designed anti-CD20xCD3 bispecific antibody is shown in Tables 7 and 8 below, and a schematic diagram of the designed anti-CD20xCD3 bispecific antibody is shown in Figure 35A.

[0149] [Table 7]

[0150] [Table 8]

[0151] (1-2) Design of anti-BCMA×CD3 bispecific antibodies (BCMA×CD3 TribsMab CLCs) A bispecific antibody corresponding to multispecific antibody 12 in Figure 5 was designed. The bispecific antibody was designed to target BCMA and CD3. The variable region (the area enclosed by the dashed line in Figure 5) had the same sequence as the variable region of pSCHLI372 (JP 2018-502062 A), a known anti-BCMA x CD3 bispecific antibody. That is, the heavy chain variable region 372VH-BCMA (specific for BCMA) and the heavy chain variable region 372VH-CD3 (specific for CD3) of pSCHLI372, as well as the light chain variable region 372VL of pSCHLI372 were used. The light chain class of pSCHLI372 is κ. Furthermore, a sequence derived from the human IgG1 class was used for the constant region. Furthermore, a peptide linker with a basic sequence of GGGGS was designed as the sequence corresponding to the peptide linker LL in Figure 5.

[0152] The correspondence between each domain of multispecific antibody 12 in Figure 5 and the designed anti-BCMAxCD3 bispecific antibody is shown in Tables 9 and 10 below, and a schematic diagram of the designed anti-BCMAxCD3 bispecific antibody is shown in Figure 35B.

[0153] [Table 9]

[0154] [Table 10]

[0155] (2) Construction of recombinant vectors for expressing bispecific antibodies Schematic diagrams of the recombinant vectors va for expressing the a-12 chain and vb for expressing the b-12 chain, which are used to express the bispecific antibodies designed in (1) above, are shown in Figures 36A and 36B. In the figures, S represents the signal sequence, and H represents the hinge region.

[0156] First, recombinant vectors va (pCAGGS-Fd1979CD20-FdCD3, pCAGGS-Fd1979CD3-FdCD20, pCAGGS-Fd372BCMA-FdCD3, pCAGGS-372FdCD3-FdBCMA) for expression of the a-12 chain were constructed. The fully synthesized pEX-A2J2-1979VH-CD20, pEX-A2J2-1979VH-CD3, pEX-A2J2-372VH-BCMA, and pEX-A2J2-372VH-CD3 were digested with the restriction enzymes Afl II and Nhe I and ligated into pCAGGS-Fd3958-Fd3178, which had been previously digested with the same enzymes, to generate four α-chain expression vector intermediates (pCAGGS-Fd1979CD20-Fd3178, pCAGGS-Fd1979CD3-Fd3178, pCAGGS-Fd372BCMA-Fd3178, and pCAGGS-Fd372CD3-Fd3178). Subsequently, four PCR products (Eco RV-1979VH-CD20-Sac I, Eco RV-1979VH-CD3-Sac I, Eco RV-372VH-BCMA-Sac I, and Eco RV-372VH-CD3-Sac I) were obtained by polymerase chain reaction (PCR) using pEX-A2J2-1979VH-CD20, pEX-A2J2-1979VH-CD3-1979VL, pEX-A2J2-372VH-BCMA, and pEX-A2J2-372VH-CD3 as templates. These four PCR products were digested with the restriction enzymes EcoRV and SacI and ligated into four a-chain expression vector intermediates that had been previously digested with the same enzymes to generate pCAGGS-Fd1979CD20-FdCD3, pCAGGS-Fd1979CD3-FdCD20, pCAGGS-Fd372BCMA-FdCD3, and pCAGGS-372FdCD3-FdBCMA.

[0157] Next, we constructed the recombinant vectors vb (pCAGGS-1979L-Fc and pCAGGS-372L-Fc) for the b-12 chain expression. pEX-A2J2-1979VH-CD3-1979VL was digested with the restriction enzymes Afl II and Nhe I and ligated to pCAGGS-128L-Fc, which had been previously digested with the same enzymes, to generate pCAGGS-1979L-FC. Next, three PCR products (EcoRI-372VL, CL(λ), and H-Fc-NotI) were amplified by PCR using pCAGGS-372-CH1-Fc, pCAGGS-HuM291LC(λ), and pCAGGS-128L-Fc as templates, respectively. These three PCR products were ligated by overlap extension to amplify EcoRI-372VL-CL(λ)-H-Fc-NotI. The final PCR product was digested with EcoRI and NotI and then ligated into pCAGGS-128L-Fc digested with the same enzymes to generate pCAGGS-372L-Fc.

[0158] (3) Preparation of transformants Human fetal kidney-derived Expi293F cells were used as hosts. All cultures were performed at 37°C, 5% CO2, and 125 rpm.

[0159] Culture in an Erlenmeyer flask containing 30 mL of HE200 medium (Gmep) until the cell count reached 3-5 × 10 6 Transfection was performed when the cell density reached 95% or higher and the viability reached 95% or higher. (R) (Thermo) to which 60 μg of plasmid (a-chain expression vector: 30 μg, b-chain expression vector: 30 μg) had been added, and 3 mL of Opti-MEM (R) Each solution was prepared by adding 240 μL of 1 mg / mL PEI-MAX (Polysciences) to the 150 × 10 cells. These two solutions were mixed and left to stand for 20 minutes, and then added to 54 mL of HE400 (Gmep). This was used as the transfection solution. 6The cell culture medium was dispensed into 50 mL sample tubes so that the cells were collected, and the tubes were centrifuged at 1,500 rpm for 5 minutes. After the culture medium was removed, the cells were suspended in the transfection solution and cultured for 20 hours.

[0160] (4) Production of bispecific antibodies Further, 150 μL of a 0.5 M aqueous solution of sodium valproate and 240 μL of a 1 M aqueous solution of sodium propionate were added, and the mixture was cultured for 6 days under the same culture conditions as those described in (3) above.

[0161] (5) Purification of bispecific antibodies (5-1) Protein A affinity purification The cells were removed from the culture medium by centrifugation at 6,000 rpm at 4°C for 10 minutes, and the culture supernatant was filtered using a 0.45 μm pore size filter (Millex-HP, Millipore). TM The culture supernatant was added to a column (column volume: 400 μL), washed with TBS, and eluted with Gentle Ag / Ab Elution Buffer (Thermo). Each fraction during the purification process was analyzed by SDS-PAGE. The eluted fraction containing the target protein was first dialyzed against TBS and then dialyzed twice against PBS (10 mM Na2PO4, 1.76 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4). The absorbance at 280 nm was measured using an absorption spectrophotometer (Nanophotometer NP80, IMPLEN), and the extinction coefficient (0.1%, 280 nm) calculated from the amino acid sequence of TribsMab CLC (BCMA × CD3 TribsMab CLC; 1.539 mg -1 mL cm -1 ,CD20×CD3 TribsMab CLC;1.471mg -1 mL cm -1The target protein was quantified using a chromatographic method. The absorbance at 320 nm was subtracted as background. Each Protein A-purified TribsMab CLC solution was loaded onto a gel filtration chromatography column, Superdex200 10 / 300GL (GE Healthcare), equilibrated with PBS. Elution was performed using an AKTA Prime plus (GE Healthcare) at a flow rate of 0.5 mL / min. The eluted fractions containing the peak absorbance at 280 nm were collected.

[0162] (5-2) Purification by cation exchange chromatography The fractions obtained in 5-1 above were purified by cation exchange chromatography in the same manner as in 5-2 of Test Example 1. Cation exchange chromatograms for the CD20×CD3 TribsMab CLC (Examples 10 and 11) and the BCMA×CD3 TribsMab CLC (Examples 12 and 13) are shown in Figure 37, and the electrophoretic analysis results of the obtained peak fractions are shown in Figure 38. As shown in Figure 37, two peaks were confirmed in each of the fractions obtained in 5-1 above. As shown in Figure 38, these two peaks were identified as the bispecific antibody of interest (the even-numbered bands in the figure) and a b-chain by-product (the odd-numbered bands in the figure), and it was found that these fractions could be separated.

[0163] The above results and the results of Test Example 1, 5-2 (Figure 27) demonstrated that the bispecific antibodies of the present invention can separate the b-chain tetramer by cation exchange chromatography, regardless of the variable region used.

[0164] Test Example 6: Evaluation of activity of multispecific antibodies (anti-CD20 × CD3 bispecific antibody, anti-BCMA × CD3 bispecific antibody) (1) Evaluation of the binding activity of anti-CD20×CD3 bispecific antibodies The anti-CD20×CD3 bispecific antibodies of Examples 10 and 11 prepared in Test Example 5 (purified by cation exchange chromatography) were evaluated for their binding activity to Raji cells (CD20-positive cells) and T-LAK cells (CD3-positive cells) as follows.

[0165] Raji cells cultured in 10% FBS / RPMI 1640 medium and T-LAK cells cultured in 5 mL of 10% FBS / RPMI 1640 medium supplemented with 1.4 μL of IL-2 (250 IU / μL) were used. As primary antibodies, solutions of the TribsMab CLCs from Examples 10 and 11 diluted with 0.1% NaN3 / PBS to a final concentration of 500 nM were used. After reacting the CD20×CD3 TribsMab CLCs and CD3×CD20 TribsMab CLCs from Examples 10 and 11 with Raji cells or T-LAK cells, 1 μL of anti-human IgG (Fc-specific)-FITC antibody (Sigma Aldrich) and 499 μL of 0.1% NaN3 / PBS were added as secondary antibodies and allowed to react. BD Accuri TM Analysis was performed using C6 (BD Biosciences).

[0166] The results are shown in Figure 39. As shown in Figure 39, peak shifts were confirmed in both Raji cells (CD20-positive cells) and T-LAK cells (CD3-positive cells) for the CD20xCD3 TribsMab CLCs of Examples 10 and 11, demonstrating binding activity to both CD20 and CD3.

[0167] (2) Evaluation of the binding activity of anti-BCMA × CD3 bispecific antibodies The anti-BCMA×CD3 bispecific antibodies of Examples 12 and 13 prepared in Test Example 5 (purified by cation exchange chromatography) were evaluated for their binding activity to T-LAK cells (CD3-positive cells) as follows.

[0168] Raji cells cultured in 10% FBS / RPMI1640 medium and T-LAK cells cultured in 5 mL of 10% FBS / RPMI-1640 medium supplemented with 1.4 μL of IL-2 (250 IU / μL) were used. The primary antibody used was a solution of TribsMab CLC from Examples 10 and 11 diluted with 0.1% NaN3 / PBS to a final concentration of 500 nM. After reacting the BCMA×CD3 TribsMab CLC from Examples 12 and 13 with T-LAK, 1 μL of anti-human IgG (Fc-specific)-FITC antibody (Sigma Aldrich) and 499 μL of 0.1% NaN3 / PBS were added as secondary antibodies and allowed to react. BD Accuri TM Analysis was performed using C6 (BD Biosciences). The results are shown in Figure 40A. As shown in Figure 40A, a peak shift was confirmed in T-LAK cells (CD3-positive cells) for the BCMA×CD3 TribsMab CLCs of Examples 12 and 13, demonstrating binding activity to CD3.

[0169] SPR analysis was performed using a Biacore3000 (GE Healthcare). PBS containing 0.005% Tween-20 (PBST) was used as the running buffer at 25°C. First, BCMA-ECD-Fc was immobilized on the surface of a CM5 sensor chip using an amine coupling kit (GE Healthcare). Next, to examine the specific binding between the antibody and antigen, BCMA-CD3 TribsMab CLC and CD3-BCMA TribsMab CLC (1 μM each) were added to the sensor chip at a flow rate of 20 μL / min for 2 minutes. 10 mM glycine-HCl (pH 1.5) was used as the regeneration reagent. The results are shown in Figure 40B. As can be seen from Figure 40B, the interaction between BCMA×CD3 TribsMab CLC and BCMA in Examples 12 and 13 was analyzed by SPR. The response increased when each antibody was added to the BCMA-ECD-Fc-immobilized sensor chip, confirming the binding activity of these antibodies to BCMA.

[0170] From the above, the BCMA×CD3 TribsMab CLCs of Examples 12 and 13 were found to have binding activity to both BCMA and CD3.

[0171] (3) Evaluation of the bispecificity of anti-CD20×CD3 bispecific antibodies The bispecificity of the CD20xCD3 TribsMab CLC was evaluated by an LDH assay using CD20-positive Raji cells (target cells) and CD3-positive T-LAK cells (effector cells). The experiment was performed at an E (effector cells) to T (target cell) ratio of 20:1. 4.0 x 10 T-LAK cells were cultured in the same manner as in (1) of this test example. 6 After suspending the cells at a concentration of 2.0 × 10 cells / mL, 25 μL was added to each well. 5 The cells were suspended in 1% FBS / RPMI1640 at a concentration of 1000 cells / mL, and then 50 μL was added per well. The antibody was diluted to a concentration of 40 nM, and 25 μL was added per well, followed by incubation for 3 hours. After incubation, the procedure followed the instructions in the Cytotoxicity LDH Assay Kit-WST (Dojindo Molecular Technologies) manual. The absorbance of each well was measured using iMark TM A Microplate Reader (BioRad) was used. At this time, the absorbance at 630 nm was subtracted as the background, and the cytotoxicity rate was calculated using the following formula.

[0172]

number

[0173] The results are shown in Figure 41A. As is clear from Figure 41A, the CD20-CD3 TribsMab CLC of Example 10 induced significantly more cytotoxicity than the monospecific antibodies, suggesting that it crosslinked CD20-positive Raji cells and CD3-positive T-LAK cells.

[0174] (4) Evaluation of the bispecificity of anti-BCMA × CD3 bispecific antibodies The bispecificity of BCMA×CD3 TribsMab CLC was evaluated by flow cytometry analysis using CD3-positive T-LAK cells and BCMA-ECD-Fc-FITC. T-LAK cells were cultured as described in (1) of this experiment, and 500 nM of BCMA×CD3 TribsMab CLC was added as the primary antibody. BCMA-ECD-Fc-FITC was then added to a final concentration of 250 nM.

[0175] The results are shown in Figure 41B. As is clear from Figure 41B, the BCMA×CD3 TribsMab CLCs of Examples 12 and 13 crosslinked fluorescently labeled BCMA-ECD-Fc and CD3-positive T-LAK cells.

[0176] [Test Example 7: Bispecific antibody with cleaved linker] The linker of the HER2×HER3 TribsMabCLC of Example 1 was cleaved with HRV3 protease to prepare linker-cleaved Her2×HER3 TribsMabCLC (Example 14).

[0177] 5 μL of Turbo3C (HRV3C) protease (Funakoshi) was added to 1 mg of HER2×HER3 TribsMabCLC (purified with Protein A) from Example 1 and allowed to stand overnight at 4°C. Next, to remove the protease, the sample was purified by column chromatography using Glutathione Sepharose 4B (GE Healthcare). After equilibration with PBS, each antibody solution was added and the flow-through was collected. The remaining protease was removed by washing the column with PBS and then eluting with elution buffer (50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0). The purified sample was analyzed by SDS-PAGE.

[0178] The results of SDS-PAGE are shown in Figure 42. As is clear from Figure 42, it was confirmed that the linker of the HER2×HER3 TribsMabCLC of Example 1 was cleaved and the bispecific antibody of Example 14 was obtained.

[0179] The binding activity of the linker-cleaved HER2×HER3 TribsMabCLC of Example 14 obtained as described above to HER2- and HER3-positive human breast adenocarcinoma cells MCF-7 was evaluated by flow cytometry.

[0180] MCF-7 cells were cultured in 10% FBS / DMEM medium. The primary antibody, linker-cleaved HER2×HER3 TribsMabCLC (500 nM) from Example 14, was incubated with the MCF-7 cells for 30 minutes, followed by washing twice with 0.1% NaN3 / PBS. Subsequently, 1 μL of anti-human IgG (Fc-specific)-FITC antibody (Sigma-Aldrich) and 499 μL of 0.1% NaN3 / PBS were added as secondary antibodies, and the incubation was continued for 30 minutes. The cells were then washed twice with 0.1% NaN3 / PBS. Flow cytometry analysis was then performed on the BD Accuri™ C6 (BD Biosciences).

[0181] The results are shown in Figure 43. As is clear from Figure 43, the linker-cleaved HER2xHER3 TribsMabCLC of Example 14 also maintained its binding activity. [Explanation of symbols]

[0182] 1,1',11,11',11'',11''',12,13,21,22,23,31,32,33...multispecific antibody a, a-11, a-12, a-13, a-21, a-22, a-23, a-31, a-2, a-33...Polypeptide a chain Va1...Valable region Va1 VHa1: Heavy chain variable region VHa1 Ca1: Constant region Ca1 CHa1…Heavy chain constant region CHa1 LL, LL-11...peptide linker LL Lr1: Protease recognition sequence Lr1 Lr2: Protease recognition sequence Lr2 Va2: Variable region Va2 VHa2: Heavy chain variable region VHa2 Ca2: Stationary Ca2 CHa2…Heavy chain constant region CHa2 b, b-1', b-12, b-13...Polypeptide b chain Vb: Variable region Vb VLb: Light chain variable region VLb Cb…constant region Cb CLb…Light chain constant region CLb ScFv1,ScFv2…single chain antibody a'...polypeptide a' chain a''...polypeptide a'' chain Lr1': Cleavage fragment Lr1' of protease recognition sequence Lr1 Lr2': Cleavage fragment Lr2' of protease recognition sequence Lr2 va: recombinant vector va vb...recombinant vector vb va'...expression vector va' vb'...expression vector vb'

Claims

1. A multispecific antibody having a Fab region comprising one polypeptide a chain as follows and two polypeptide b chains as follows: a polypeptide a chain comprising a polypeptide in which a variable region Va1, a constant region Ca1 (excluding the region linked to the Fc domain), a peptide linker LL, a variable region Va2, and a constant region Ca2 are linked in this order; and A polypeptide b chain comprising a polypeptide in which a variable region Vb is linked to a constant region Cb that binds to the constant region Ca1 or the constant region Ca2.

2. The multispecific antibody of claim 1, wherein the length of the peptide linker LL is 70 to 280 Å.

3. The multispecific antibody of claim 1 or 2, wherein the peptide linker LL comprises a protease recognition sequence.

4. The multispecific antibody according to claim 3 , wherein the peptide linker LL comprises a protease recognition sequence Lr1 on the constant region Ca1 side and a protease recognition sequence Lr2 on the variable region Va2 side.

5. IgD, IgE, IgG, or F(ab') 2 The multispecific antibody according to any one of claims 1 to 4,

6. The multispecific antibody according to any one of claims 1 to 5, wherein the polypeptide a chain comprises a polypeptide in which a heavy chain variable region VHa1, a heavy chain constant region CHa1, a peptide linker LL, a heavy chain variable region VHa2, and a heavy chain constant region CHa2 are linked in this order.

7. The multispecific antibody according to any one of claims 1 to 5, wherein the polypeptide a chain comprises a polypeptide in which a heavy chain variable region VHa1, a light chain constant region CLa1, a peptide linker LL, a heavy chain variable region VHa2, and a light chain constant region CLa2 are linked in this order.

8. The multispecific antibody according to any one of claims 1 to 7, wherein a single-chain antibody is further bound to the variable region Va1 and / or the constant region Ca2.

9. A multispecific antibody having a Fab region comprising one polypeptide a' chain as follows: one polypeptide a'' chain as follows: and two polypeptide b chains as follows: a polypeptide a' chain comprising a polypeptide in which a variable region Va1, a constant region Ca1 (excluding the region linked to the Fc domain), and a cleavage fragment Lr1' of a protease recognition sequence Lr1 are linked in this order; A polypeptide a'' chain comprising a polypeptide in which a cleavage fragment Lr2' of the protease recognition sequence Lr2, a variable region Va2, and a constant region Ca2 are linked in this order; and A polypeptide b chain comprising a polypeptide in which a variable region Vb is linked to a constant region Cb that binds to the constant region Ca1 or the constant region Ca2.

10. a recombinant vector va containing DNA encoding a polypeptide a chain comprising a polypeptide in which a variable region Va1, a constant region Ca1 (excluding the region linked to the Fc domain), a peptide linker LL, a variable region Va2, and a constant region Ca2 are linked in this order; a recombinant vector vb containing DNA encoding a polypeptide b chain comprising a polypeptide in which a variable region Vb and a constant region Cb that binds to the constant region Ca1 or the constant region Ca2 are linked; A transformant obtained by transforming a host with the

11. a recombinant vector va containing DNA encoding a polypeptide a chain comprising a polypeptide in which a variable region Va1, a constant region Ca1 (excluding the region linked to the Fc domain), a peptide linker LL, a variable region Va2, and a constant region Ca2 are linked in this order; a recombinant vector vb containing DNA encoding a polypeptide b chain comprising a polypeptide in which a variable region Vb and a constant region Cb that binds to the constant region Ca1 or the constant region Ca2 are linked; A method for producing a multispecific antibody, comprising an antibody production step of transforming a host with the above-mentioned vector and culturing the resulting transformant.

12. the peptide linker LL comprises a protease recognition sequence Lr1 on the constant region Ca1 side and a protease recognition sequence Lr2 on the variable region Va2 side; The method for producing a multispecific antibody according to claim 11, further comprising, after the antibody production step, a linker cleavage step of cleaving the peptide linker LL in the produced antibody using a protease corresponding to the protease recognition sequence Lr1 and the protease recognition sequence Lr2.

13. an expression vector va' comprising, in this order, a cloning site CS1 for incorporating the variable region Va1, DNA encoding the constant region Ca1 (excluding the region linked to the Fc domain), DNA encoding the peptide linker LL, a cloning site CS2 for incorporating the variable region Va2, and DNA encoding the constant region Ca2; an expression vector vb' comprising a cloning site CS for inserting a variable region Vb and DNA encoding a constant region Cb that binds to the constant region Ca1 or the constant region Ca2; A kit for producing a multispecific antibody, comprising:

14. A diagnostic agent comprising the multispecific antibody according to any one of claims 1 to 9.

15. A pharmaceutical composition comprising the multispecific antibody of any one of claims 1 to 9.

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