Method for quantifying mispaired light chain exchanger of bispecific antibody

Abstract

According to the present invention, by using an antibody that recognizes a light chain exchanger of a mispaired variant of a bispecific antibody, it became possible to accurately quantify or detect a light chain exchanger contained as an impurity when preparing a bispecific antibody.

Description

Method for quantifying mispaired light chain exchangers in bispecific antibodies

[0001] The present disclosure relates to a method for detecting mispaired light chain exchangers in multispecific antibodies and antibodies that recognize mispaired light chain exchangers. In one aspect, the present disclosure relates to a method for quantitatively detecting light chain exchangers in bispecific antibodies and antibodies that recognize light chain exchangers.

[0002] Bispecific antibodies (BsAbs) are antibodies that specifically bind to two different antigens (epitopes). Because they can exert mechanisms of action that cannot be achieved with conventional monospecific antibodies, they are being developed as next-generation antibody drugs. However, the preparation of BsAbs in a single cell is difficult because random association of heavy and light chains results in the generation of up to nine mispairs in addition to the desired BsAb. To address this issue, engineering methods have been developed to control the association between heavy chains and heavy chains and between heavy chains and light chains.

[0003] Typical techniques for controlling heavy-heavy chain interactions include knobs-into-holes and charge modification (Non-Patent Document 1, Non-Patent Document 1). Known techniques for controlling heavy-light chain interactions include orthogonal Fab (Non-Patent Document 2), which utilizes steric complementarity and charge modification, and CrossMab (Non-Patent Document 3), which crosses over heavy-light chain domains. FAST-Ig (Non-Patent Document 4) is an example of an orthogonal Fab that utilizes charge modification. These techniques have made it possible to prepare highly pure BsAbs.

[0004] However, quality control of antibody drugs requires a method for accurately measuring the content of each mispair. Methods for separating and quantifying mispairs include chromatography, which utilizes differences in pI, and LC-MS, which utilizes differences in molecular weight of mispairs (Patent Document 2, Non-Patent Document 5). However, these methods make it difficult to analyze "light chain exchangers" in which two light chains have been exchanged. Because the theoretical pI and molecular weight of the target BsAb and the light chain exchanger are identical, these methods cannot distinguish them.

[0005] A method for quantifying light chain exchangers has been reported in which the amount of mispairs resulting from the target BsAb and the common light chain is estimated from the association probability of the heavy and light chains (Non-Patent Document 5), but this is an estimate and does not allow for absolute quantification. Another method has been reported in which purified BsAb is enzymatically digested at the hinge region to quantify the Fab as a single entity based on the difference in Fab mass or pI (Patent Document 3), but this is difficult to analyze when the masses and pIs of the heavy and light chains of each entity are similar.

[0006] WO2006106905WO2007114325WO2021201202

[0007] Nat Biotechnol. 1998 Jul;16(7):677-81.Nat Biotechnol. 2014 Feb;32(2):191-8.Proc Natl Acad Sci US A. 2011 Jul 5;108(27):11187-92.MAbs. 2023 Jan-Dec;15(1):2222441.MAbs. 2016 Nov / Dec;8(8):1467-1476.

[0008] The present invention has been made in view of the above circumstances, and an object thereof is to provide a method for quantifying light chain exchange products that can be used equally for various variable region sequences.

[0009] The present invention provides a method for accurately quantifying light chain exchangers using antibodies that recognize the heavy-light chain interface with modifications to promote correct heavy-light chain association, which overcomes the limitations of conventional techniques and is equally applicable to a wide range of variable region sequences.

[0010] The present disclosure specifically encompasses the following exemplary embodiments: [A-1] A method for quantifying or detecting mispaired variants of a multispecific antigen-binding molecule, wherein the mispaired variants comprise at least two different types of F(ab) that are not present in the multispecific antigen-binding molecule, the method comprising: (a) contacting a first detection antibody with a sample containing the mispaired variants, (b) contacting a second detection antibody with the mispaired variant bound to the first detection antibody, and (c) quantifying or detecting the second detection antibody bound to the mispaired variant, wherein one of the first or second detection antibodies binds to one of the F(ab), and the other binds to the other F(ab). [A-2] The mispair variant comprises a first and a second F(ab), wherein the first F(ab) has at least one first pair of amino acid residues forming a VH-VL interface or a CH1-CL interface, and both amino acid residues forming the first pair of amino acid residues are positively charged, and the second F(ab) has at least one second pair of amino acid residues forming a VH-VL interface or a CH1-CL interface, and both amino acid residues forming the second pair of amino acid residues are negatively charged, and one of the first or second detection antibodies binds to one of the first or second F(ab), and the other binds to the other of the first or second F(ab). [A-3] The method according to [A-1], wherein the mispair variants include: (1) a first and a second F(ab) in which the charge of the amino acids at positions 147 and 175 in CH1 and the amino acids at positions 131 and 160 in CL is controlled to repel each other; (2) a first and a second F(ab) in which the charge of the amino acids at positions 147 and 175 in CH1 and the amino acids at positions 131 and 180 in CL is controlled to repel each other; or (3) a first and a second F(ab) in which the charge of the amino acids at positions 147 and 175 in CH1 and the amino acids at positions 131, 160, and 180 in CL is controlled to repel each other;Here, the position numbers of CH1 follow EU numbering, and the position numbers of CL follow Kabat numbering. [A-4] The method according to [A-1], wherein the mispair variant F(ab) comprises the following amino acid residues: (1) K at positions 147 and 175 in CH1; and K at positions 131 and 160 in CL; (2) K at positions 147 and 175 in CH1; and K at positions 131 and 180 in CL; (3) K at positions 147 and 175 in CH1; and K at positions 160 and 180 in CL; (4) K at positions 147 and 213 in CH1; and K at positions 123 and 131 in CL; (5) K at positions 147 and 213 in CH1; and K at positions 123 and 160 in CL; (6) K at positions 147 and 213 in CH1; (7) K at positions 175 and 213 in CH1; and K at positions 123 and 131 in CL; (8) K at positions 175 and 213 in CH1; and K at positions 123 and 160 in CL; (9) K at positions 175 and 213 in CH1; and K at positions 123 and 180 in CL; (10) K at positions 147 and 175 in CH1; and K at positions 131, 160, and 180 in CL; (11) K at positions 147, 175, and 213 in CH1; and K at positions 123, 131, 160, and 180 in CL; wherein the position numbers of CH1 are according to EU numbering, and the position numbers of CL are according to Kabat numbering. [A-5] The method according to [A-1], wherein the mispaired variant F(ab) contains the following amino acid residues: (1) E at positions 147 and 175 in CH1; and E at positions 131 and 160 in CL; (2) E at positions 147 and 175 in CH1; and E at positions 131 and 180 in CL; (3) E at positions 147 and 175 in CH1; and E at positions 160 and 180 in CL; (4) E at positions 147 and 213 in CH1; and E at positions 123 and 131 in CL;(5) E at positions 147 and 213 in CH1; and E at positions 123 and 160 in CL; (6) E at positions 147 and 213 in CH1; and E at positions 123 and 180 in CL; (7) E at positions 175 and 213 in CH1; and E at positions 123 and 131 in CL; (8) E at positions 175 and 213 in CH1; and E at positions 123 and 160 in CL; (9) E at positions 175 and 213 in CH1; and E at positions 123 and 180 in CL; (10) E at positions 147 and 175 in CH1; and E at positions 131, 160, and 180 in CL; (11) E at positions 147, 175, and 213 in CH1; and E at positions 123, 131, 160, and 180 in CL; wherein the position numbers in CH1 are according to EU numbering, and the position numbers in CL are according to Kabat numbering. [A-6] The method according to [A-1], wherein the first detection antibody is biotinylated. [A-7] The method according to [A-1], wherein the second detection antibody is fluorescently labeled. [A-8] An antibody that binds to an F(ab) present in a mispaired variant of a multispecific antigen-binding molecule but does not bind to an F(ab) present in the multispecific antigen-binding molecule. [A-9] The antibody according to [A-8], wherein the F(ab) of the mispaired variant has at least one pair of amino acid residues forming a VH-VL interface or a CH1-CL interface, and both amino acid residues forming the amino acid residue pair have the same charge. [A-10] The antibody according to [A-8], which binds to the following F(ab): (1) an F(ab) in which the charge is controlled so that the amino acids at positions 147 and 175 in CH1 and the amino acids at positions 131 and 160 in CL repel each other; (2) an F(ab) in which the charge is controlled so that the amino acids at positions 147 and 175 in CH1 and the amino acids at positions 131 and 180 in CL repel each other; or (3) an F(ab) in which the charge is controlled so that the amino acids at positions 147 and 175 in CH1 and the amino acids at positions 131, 160, and 180 in CL repel each other;Here, the position numbers of CH1 follow EU numbering, and the position numbers of CL follow Kabat numbering. [A-11] The antibody according to [A-8], which binds to F(ab) containing the following amino acid residues present in the mispair variant: (1) K at positions 147 and 175 in CH1; and K at positions 131 and 160 in CL; (2) K at positions 147 and 175 in CH1; and K at positions 131 and 180 in CL; (3) K at positions 147 and 175 in CH1; and K at positions 160 and 180 in CL; (4) K at positions 147 and 213 in CH1; and K at positions 123 and 131 in CL; (5) K at positions 147 and 213 in CH1; and K at positions 123 and 160 in CL; (6) K at positions 147 and 213 in CH1; (7) K at positions 175 and 213 in CH1; and K at positions 123 and 131 in CL; (8) K at positions 175 and 213 in CH1; and K at positions 123 and 160 in CL; (9) K at positions 175 and 213 in CH1; and K at positions 123 and 180 in CL; (10) K at positions 147 and 175 in CH1; and K at positions 131, 160, and 180 in CL; (11) K at positions 147, 175, and 213 in CH1; and K at positions 123, 131, 160, and 180 in CL; wherein the position numbers for CH1 are according to EU numbering, and the position numbers for CL are according to Kabat numbering. [A-12] The antibody according to [A-8], selected from the following: (1) an antibody capable of binding to an F(ab) having K at positions 147 and 175 in CH1 and K at positions 131 and 160 in CL, but an F(ab) having K at positions 147 and 175 in CH1 and E at positions 131 and 160 in CL; or an antibody not capable of binding to an F(ab) having E at positions 147 and 174 in CH1 and K at positions 131 and 160 in CL;(2) An antibody capable of binding to F(ab) in which positions 147 and 175 in CH1 are K and positions 131 and 180 in CL are K, but an F(ab) in which positions 147 and 175 in CH1 are K and positions 131 and 180 in CL are E; or an antibody incapable of binding to F(ab) in which positions 147 and 175 in CH1 are E and positions 131 and 180 in CL are K; (3) An antibody capable of binding to F(ab) in which positions 147 and 175 in CH1 are K and positions 160 and 180 in CL are K, but an F(ab) in which positions 147 and 175 in CH1 are K and positions 160 and 180 in CL are E; or an antibody that cannot bind to an F(ab) in which positions 147 and 175 in CH1 are E and positions 160 and 180 in CL are K; (4) an antibody that can bind to an F(ab) in which positions 147 and 213 in CH1 are K and positions 123 and 131 in CL are K, but an F(ab) in which positions 147 and 213 in CH1 are K and positions 123 and 131 in CL are E; or an antibody that cannot bind to an F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 131 in CL are K; (5) an antibody that can bind to an F(ab) in which positions 147 and 213 in CH1 are K and positions 123 and 160 in CL, but (6) An antibody that can bind to an F(ab) in which positions 147 and 213 in CH1 are K and positions 123 and 160 in CL are E; or an antibody that cannot bind to an F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 160 in CL are K; (7) An antibody that can bind to an F(ab) in which positions 147 and 213 in CH1 are K and positions 123 and 180 in CL are K, but an F(ab) in which positions 147 and 213 in CH1 are K and positions 123 and 180 in CL are E;or an antibody that cannot bind to F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 180 in CL are K; (7) an antibody that can bind to F(ab) in which positions 175 and 213 in CH1 are K and positions 123 and 131 in CL are K, but an F(ab) in which positions 175 and 213 in CH1 are K and positions 123 and 131 in CL are E; or an antibody that cannot bind to F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 131 in CL are K; (8) an antibody that can bind to F(ab) in which positions 175 and 213 in CH1 are K and positions 123 and 160 in CL, but (9) An antibody that can bind to an F(ab) in which positions 175 and 213 in CH1 are K and positions 123 and 131 in CL are K; or an antibody that cannot bind to an F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 131 in CL are K; (10) An antibody that can bind to an F(ab) in which positions 175 and 213 in CH1 are K and positions 123 and 180 in CL are K, but cannot bind to an F(ab) in which positions 175 and 213 in CH1 are K and positions 123 and 180 in CL are K; or an antibody that cannot bind to an F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 180 in CL; (10) An antibody capable of binding to F(ab) in which positions 147 and 175 in CH1 are K and positions 131, 160, and 180 in CL are K, but an F(ab) in which positions 147 and 175 in CH1 are K and positions 131, 160, and 180 in CL are E; or an antibody incapable of binding to F(ab) in which positions 147 and 175 in CH1 are E and positions 131, 160, and 180 in CL are K; or (11) An antibody capable of binding to F(ab) in which positions 147, 175, and 213 in CH1 are K and positions 123, 131, 160, and 180 in CL are K, but F(ab) in which K is at positions 147, 175, and 213 in CH1 and E is at positions 123, 131, 160, and 180 in CL;or an antibody that cannot bind to F(ab)2 in which positions 147, 175, and 213 in CH1 are E and positions 123, 131, 160, and 180 in CL are K; wherein the position numbers in CH1 are according to EU numbering, and the position numbers in CL are according to Kabat numbering. [A-13] The antibody according to [A-8], which binds to F(ab) containing the following amino acid residues present in the mispair variant: (1) E at positions 147 and 175 in CH1; and E at positions 131 and 160 in CL; (2) E at positions 147 and 175 in CH1; and E at positions 131 and 180 in CL; (3) E at positions 147 and 175 in CH1; and E at positions 160 and 180 in CL; (4) E at positions 147 and 213 in CH1; and E at positions 123 and 131 in CL; (5) E at positions 147 and 213 in CH1; and E at positions 123 and 160 in CL; (6) E at positions 147 and 213 in CH1; and E at positions 123 and 180 in CL; (7) E at positions 175 and 213 in CH1; and E at positions 123 and 131 in CL; (8) E at positions 175 and 213 in CH1; and E at positions 123 and 160 in CL; (9) E at positions 175 and 213 in CH1; and E at positions 123 and 180 in CL; (10) E at positions 147 and 175 in CH1; and E at positions 131, 160, and 180 in CL; (11) E at positions 147, 175, and 213 in CH1; and E at positions 123, 131, 160, and 180 in CL; wherein the position numbers of CH1 are according to EU numbering, and the position numbers of CL are according to Kabat numbering. [A-14] The antibody according to [A-8], selected from the following: (1) an antibody capable of binding to F(ab) having E at positions 147 and 175 in CH1; and E at positions 131 and 160 in CL, but having K at positions 147 and 175 in CH1; and E at positions 131 and 160 in CL;or an antibody that does not bind to F(ab) in which positions 147 and 174 in CH1 are E and positions 131 and 160 in CL are K; (2) an antibody that can bind to F(ab) in which positions 147 and 175 in CH1 are E and positions 131 and 180 in CL are E, but an F(ab) in which positions 147 and 175 in CH1 are K and positions 131 and 180 in CL are E; or an antibody that cannot bind to F(ab) in which positions 147 and 175 in CH1 are E and positions 131 and 180 in CL; (3) an antibody that can bind to F(ab) in which positions 147 and 175 in CH1 are E and positions 160 and 180 in CL are E, but (4) An antibody that can bind to an F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 131 in CL are E, but cannot bind to an F(ab) in which positions 147 and 213 in CH1 are K and positions 123 and 131 in CL are E; or an antibody that cannot bind to an F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 131 in CL are K; or an antibody that cannot bind to an F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 131 in CL are K; (5) An antibody capable of binding to F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 160 in CL are E, but in which positions 147 and 213 in CH1 are K and positions 123 and 160 in CL are E; or an antibody incapable of binding to F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 160 in CL are K; (6) An antibody capable of binding to F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 180 in CL are E, but in which positions 147 and 213 in CH1 are K and positions 123 and 180 in CL are E;or an antibody that cannot bind to F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 180 in CL are K; (7) an antibody that can bind to F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 131 in CL are E, but an F(ab) in which positions 175 and 213 in CH1 are K and positions 123 and 131 in CL are E; or an antibody that cannot bind to F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 131 in CL are K; (8) an antibody that can bind to F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 131 in CL are E, but (9) An antibody that can bind to an F(ab) in which positions 175 and 213 in CH1 are K and positions 123 and 131 in CL are E; or an antibody that cannot bind to an F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 131 in CL are K; (10) An antibody that can bind to an F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 180 in CL are E, but an F(ab) in which positions 175 and 213 in CH1 are K and positions 123 and 180 in CL are E; or an antibody that cannot bind to an F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 180 in CL are K; (10) An antibody capable of binding to F(ab) in which positions 147 and 175 in CH1 are E and positions 131, 160, and 180 in CL are E, but an F(ab) in which positions 147 and 175 in CH1 are K and positions 131, 160, and 180 in CL are E; or an antibody incapable of binding to F(ab) in which positions 147 and 175 in CH1 are E and positions 131, 160, and 180 in CL are K; or (11) An antibody capable of binding to F(ab) in which positions 147, 175, and 213 in CH1 are E and positions 123, 131, 160, and 180 in CL are E, but F(ab) in which positions 147, 175, and 213 in CH1 and positions 123, 131, 160, and 180 in CL are E;or an antibody that cannot bind to F(ab)2 in which positions 147, 175, and 213 in CH1 are E and positions 123, 131, 160, and 180 in CL are K; wherein the position numbers in CH1 are according to EU numbering, and the position numbers in CL are according to Kabat numbering. [A-15] The antibody according to [A-8], comprising the following VH and VL: (1) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 64, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 69, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 75; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 80, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 90; (2) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 70, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 76; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 81, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 91; (3) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 70, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 76; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 86, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 91; (4) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 70, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 76; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 92;(5) VH comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 70, and HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 77; and VL comprising LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 92; (6) VH comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 70, and HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 77; and VL comprising LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 86, and LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 92; (7) VH comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 70, and HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 77; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 93; (8) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 70, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 77; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 81, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 92; (9) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 66, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 71, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 77; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 87, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 94;(10) VH comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 66, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 71, and HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 77; and VL comprising LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 93; (11) VH comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 72, and HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 78; and VL comprising LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 93; (12) VH comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 71, and HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 78; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 93; (13) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 66, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 71, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 78; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 93; (14) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 66, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 73, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 77; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 93;(15) VH comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 67, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 74, and HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 79; and VL comprising LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 83, LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 88, and LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 95; (16) VH comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 67, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 74, and HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 79; and VL comprising LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 84, LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 88, and LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 95; (17) VH comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 67, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 74, and HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 79; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 83, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 89, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 95; (18) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 68, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 74, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 79; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 84, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 88, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 95; or (19) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 68, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 74, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 79;and a VL comprising LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 83, LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 89, and LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 96. [A-16] The antibody according to [A-8], comprising the following VH and VL: (1) a VH comprising the amino acid sequence set forth in SEQ ID NO: 8 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 31; (2) a VH comprising the amino acid sequence set forth in SEQ ID NO: 8 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 32; (3) a VH comprising the amino acid sequence set forth in SEQ ID NO: 8 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 33; (4) a VH comprising the amino acid sequence set forth in SEQ ID NO: 9 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 33; (5) a VH comprising the amino acid sequence set forth in SEQ ID NO: 9 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 34; (6) a VH comprising the amino acid sequence set forth in SEQ ID NO: 9 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (7) a VH comprising the amino acid sequence set forth in SEQ ID NO: 9 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 36; (8) a VH comprising the amino acid sequence set forth in SEQ ID NO: 10 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 37; (9) (10) VH comprising the amino acid sequence set forth in SEQ ID NO: 10 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (10) VH comprising the amino acid sequence set forth in SEQ ID NO: 11 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (11) VH comprising the amino acid sequence set forth in SEQ ID NO: 12 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (12) VH comprising the amino acid sequence set forth in SEQ ID NO: 13 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (13) VH comprising the amino acid sequence set forth in SEQ ID NO: 14 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (14) VH comprising the amino acid sequence set forth in SEQ ID NO: 9 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (15) VH comprising the amino acid sequence set forth in SEQ ID NO: 10 and VL comprising the amino acid sequence set forth in SEQ ID NO: 37;(16) VH comprising the amino acid sequence set forth in SEQ ID NO: 10 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (17) VH comprising the amino acid sequence set forth in SEQ ID NO: 11 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (18) VH comprising the amino acid sequence set forth in SEQ ID NO: 12 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (19) VH comprising the amino acid sequence set forth in SEQ ID NO: 13 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (20) VH comprising the amino acid sequence set forth in SEQ ID NO: 14 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (21) VH comprising the amino acid sequence set forth in SEQ ID NO: 9 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (22) VH comprising the amino acid sequence set forth in SEQ ID NO: 10 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (23) VH comprising the amino acid sequence set forth in SEQ ID NO: 11 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (24) VH comprising the amino acid sequence set forth in SEQ ID NO: 12 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (25) VH comprising the amino acid sequence set forth in SEQ ID NO: 13 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (26) VH comprising the amino acid sequence set forth in SEQ ID NO: 14 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (27) VH comprising the amino acid sequence set forth in SEQ ID NO: 7 and VL comprising the amino acid sequence set forth in SEQ ID NO: 38; (28) VH comprising the amino acid sequence set forth in SEQ ID NO: 7 and VL comprising the amino acid sequence set forth in SEQ ID NO: 39; (29) VH comprising the amino acid sequence set forth in SEQ ID NO: 15 and VL comprising the amino acid sequence set forth in SEQ ID NO: 38; (30) VH comprising the amino acid sequence set forth in SEQ ID NO: 15 and VL comprising the amino acid sequence set forth in SEQ ID NO: 40; (31) VH comprising the amino acid sequence set forth in SEQ ID NO: 7 and VL comprising the amino acid sequence set forth in SEQ ID NO: 30; (32) VH comprising the amino acid sequence set forth in SEQ ID NO:6 and VL comprising the amino acid sequence set forth in SEQ ID NO:29;or (33) A VH comprising the amino acid sequence set forth in SEQ ID NO: 7, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 30. [A-17] A nucleic acid encoding the antibody or antigen-binding fragment thereof set forth in any of [A-8] to [A-16]. [A-18] A host cell harboring the nucleic acid set forth in [A-17]. [A-19] A method for producing an antibody for detecting mispaired variants, comprising a step of immunizing with F(ab) present in a mispaired variant of a multispecific antigen-binding molecule, wherein the F(ab) has at least one pair of amino acid residues forming a VH-VL interface or a CH1-CL interface, and both amino acid residues forming the amino acid residue pair have the same charge.;

[0011] [B-1] A method for quantifying mispaired antibodies in a mixture of bispecific antibodies and their mispaired by-products, wherein the bispecific antibody comprises heavy chain A, heavy chain B, light chain A, and light chain B, and comprises heavy chain A associated with light chain A and heavy chain B associated with light chain B; and the mispaired antibody comprises heavy chain A, heavy chain B, light chain A, and light chain B, and comprises heavy chain A associated with light chain B (first mispaired heavy chain A-light chain B) and heavy chain B associated with light chain A (second mispaired heavy chain A-light chain B), the method comprising: (a) contacting the mixture of the bispecific antibody and its mispaired antibody by-products with a first detection antibody; (b) contacting the antibody bound to the first detection antibody in step (a) with a second detection antibody; (c) measuring the amount of binding of the second detection antibody to the antibody bound to the first detection antibody in step (b); Here, one of the first and second detection antibodies is capable of binding to an aggregate of heavy chain A and light chain B of the mispaired antibody (first mispaired heavy chain A-light chain B), and the other of the first and second detection antibodies is capable of binding to an aggregate of heavy chain B and light chain A of the mispaired antibody (second mispaired heavy chain B-light chain A). [B-2] The method according to [B-1], wherein the first or second detection antibody exhibits strong binding to the aggregate of heavy chain A and light chain B (mispaired heavy chain A-light chain B) and / or the aggregate of heavy chain B and light chain A (mispaired heavy chain B-light chain A). [B-3] The method of [B-1] or [B-2], wherein the first or second detection antibody exhibits stronger binding to the complex of heavy chain A and light chain B (mispaired heavy chain A-light chain B) and / or the complex of heavy chain B and light chain A (mispaired heavy chain B-light chain A) than to the complex of the correct pair of heavy chain A and light chain A and / or the correct complex of heavy chain B and light chain B. [B-4] The method of any of [B-1] to [B-3], wherein the first and / or second detection antibody does not substantially bind to the complex of the correct pair of heavy chain A and light chain A and / or the correct complex of heavy chain B and light chain B. [B-5] The method of any of [B-1] to [B-4], wherein the first detection antibody is a biotinylated antibody. [B-6] The method of any of [B-1] to [B-5], wherein the second detection antibody is conjugated to a fluorescent substrate.[B-7] The method according to any one of [B-1] to [B-6], wherein the heavy chain A and the light chain A each contain an amino acid residue that promotes the association between heavy chain A and light chain A. [B-8] The method according to any one of [B-1] to [B-7], wherein the heavy chain B and the light chain B each contain an amino acid residue that promotes the association between heavy chain B and light chain B.

[0012] [C-1] A method for quantifying mispaired by-product antibodies in a mixture of a bispecific antibody and its mispaired by-product antibodies, wherein the bispecific antibody comprises heavy chain A, heavy chain B, light chain A, and light chain B, and also comprises an aggregate of light chain A and heavy chain A (first correct pair: heavy chain A-light chain A) and an aggregate of light chain B and heavy chain B (second correct pair: heavy chain B-light chain B), and wherein the mispaired by-product antibodies comprise an aggregate of heavy chain A and light chain B (first mispaired heavy chain A-light chain B) and / or an aggregate of heavy chain B and light chain A (second mispaired heavy chain A-light chain B), the method comprising: (a) contacting the mixture of the bispecific antibody and its mispaired by-products with a detection antibody, (b) a step of measuring the amount of the detection antibody binding to the mispaired by-product antibody of step (a); wherein the detection antibody is capable of binding to the complex of the heavy chain A and the light chain B (first mispaired heavy chain A-light chain B) or the complex of the heavy chain B and the light chain A (second mispaired heavy chain B-light chain A), and the detection antibody does not substantially bind to the complex of the heavy chain A and the light chain A (first correct complex of heavy chain A-light chain A) or the complex of the heavy chain B and the light chain B (second correct complex of heavy chain B-light chain B); and [C-2] The method according to [C-1], comprising the following steps: (a) contacting a mixture of the bispecific antibody and its mispaired by-product with a first detection antibody; (b) contacting the antibody bound to the first detection antibody in step (a) with a second detection antibody; and (c) measuring the amount of binding of the second detection antibody to the antibody bound to the first detection antibody in step (b), wherein one of the first and second detection antibodies is capable of binding to an aggregate of heavy chain A and light chain B of the mispaired antibody (first mispaired heavy chain A-light chain B), and the other of the first and second detection antibodies is capable of binding to an aggregate of heavy chain B and light chain A of the mispaired antibody (second mispaired heavy chain B-light chain A).

[0013] [D-1] A method for quantifying / detecting mispaired variants (mutants) of a bispecific antibody, wherein the mispaired variant contains two F(ab)s that are not present in the bispecific antibody, and the two F(ab)s are different from each other. [D-2] The method described in [D-1], comprising the following steps: (a) contacting a sample (or solution) containing the mispaired variant with a first detection antibody; (b) contacting the mispaired variant bound to the first detection antibody in step (a) with a second detection antibody; and (c) detecting the amount of the second detection antibody (quantifying or detecting binding of the second detection antibody to the mispaired variant in step (b)); wherein one of the first detection antibody and the second detection antibody is capable of binding to one of the two F(ab)s contained in the mispaired variant, and the first detection antibody and the second detection antibody are capable of binding to the other of the two F(ab)s contained in the mispaired variant. [D-3] A method for quantifying or detecting mispaired variants of a bispecific antibody, wherein the mispaired variants include: a first F(ab) comprising at least one pair of amino acid residues that form a VH-VL interface or a CH1-CL interface, and wherein both of said at least one pair of amino acid residues have a positive charge; and a second F(ab) comprising at least one pair of amino acid residues that form a VH-VL interface or a CH1-CL interface, and wherein both of said at least one pair of amino acid residues have a negative charge. [D-4] The method according to [D-3], wherein neither the first nor the second F(ab) is present in a bispecific antibody (an antibody having the correct pair). [D-5] A method according to [D-3] or [D-4], comprising the following steps: (a) contacting a sample (or solution) containing a mispaired variant of a bispecific antibody with a first detection antibody; (b) contacting the mispaired variant bound to the first detection antibody of step (a) with a second detection antibody; (c) detecting the amount of the second detection antibody (quantifying or detecting the binding of the second detection antibody to the mispaired variant of step (b)); wherein one of the first detection antibody and the second detection antibody is capable of binding to one of the first and second F(ab), and the other of the first detection antibody and the second detection antibody is capable of binding to the other of the first and second F(ab).

[0014] [E-1] A method for quantifying / detecting mispaired variants (mutants) of a bispecific antibody, comprising: (a) contacting a sample (or solution) containing mispaired variants with a first detection antibody; (b) contacting the mispaired variants bound to the first detection antibody in step (a) with a second detection antibody; and (c) quantifying or detecting binding of the second detection antibody to the mispaired variants bound to the first detection antibody in step (b), wherein the mispaired variants are: (1) first and second F(ab)s in which the charge is controlled so that the amino acids at positions 147 and 175 in CH1 and the amino acids at positions 131 and 160 in CL repel each other; (2) first and second F(ab)s in which the charge is controlled so that the amino acids at positions 147 and 175 in CH1 and the amino acids at positions 131 and 180 in CL repel each other; or (3) The antibody comprises first and second F(ab)2s whose charges are controlled so that the amino acids at positions 147 and 175 in CH1 and positions 131, 160, and 180 in CL are repulsive to each other, wherein the position numbers of CH1 are according to EU numbering, and the position numbers of CL are according to Kabat numbering. [E-2] The method according to [E-1], wherein: (1) in (1) of [E-1], K is present at positions 147 and 175 in CH1; and K is present at positions 131 and 160 in CL; or E is present at positions 147 and 175 in CH1; and E is present at positions 131 and 160 in CL; (2) in (2) of [E-1], K is present at positions 147 and 175 in CH1; and K is present at positions 131 and 180 in CL; or E is present at positions 147 and 175 in CH1; and E is present at positions 131 and 180 in CL; (3) in (3) of [E-1], K is present at positions 147 and 175 in CH1; and K is present at positions 131, 160, and 180 in CL; or Positions 147 and 175 in CH1 are E; and positions 131, 160, and 180 in CL are E.[E-3] The method described in [E-1] or [E-2], wherein the bispecific antibody having the correct pair comprises: (1) a first and a second F(ab) in which positions 147 and 175 in CH1 and positions 131 and 160 in CL are charge-controlled to attract each other; (2) a first and a second F(ab) in which positions 147 and 175 in CH1 and positions 131 and 180 in CL are charge-controlled to attract each other; (3) a first and a second F(ab) in which positions 147 and 175 in CH1 and positions 131, 160, and 180 in CL are charge-controlled to attract each other. [E-4] The method according to any one of [E-1] to [E-3], wherein the first and / or second antibody is an antibody that recognizes: (1) an F(ab) comprising at least one pair of amino acid residues that form a VH-VL interface or a CH1-CL interface, and wherein both of said at least one pair of amino acid residues have a positive charge; or (2) an F(ab) comprising at least one pair of amino acid residues that form a VH-VL interface or a CH1-CL interface, and wherein both of said at least one pair of amino acid residues have a negative charge.

[0015] [F-1] A method for quantifying or detecting an antibody comprising an F(ab) that contains at least one pair of amino acid residues that form a VH-VL interface or a CH1-CL interface, and wherein each of the at least one pair of amino acid residues has the same charge. [F-2] The method described in [F-1], comprising the following steps: (a) contacting a sample (or solution) containing an antibody comprising the F(ab) with one or more detection antibodies; (b) detecting the amount of detection antibody in step (a) (quantifying or detecting binding of the detection antibody to the antibody comprising the F(ab)); wherein one of the detection antibodies can bind to the F(ab) but cannot bind to an F(ab) that contains an amino acid residue with a different type of charge at the corresponding position.

[0016] [G-1] A method for detecting an antibody, comprising: (a) contacting a detection antibody with a sample containing an antibody in which all amino acid residues at the CH1-CL association interface have side chains with the same type of charge; and (b) detecting the amount of detection antibody bound in step (a). [G-2] The method according to [G-1], in which the detection antibody specifically binds to a portion of the antibody comprising the interface between CH1 and CL.

[0017] [H-1] An antibody that binds to an F(ab) present in a mispaired variant (mutant) of a multispecific antigen-binding molecule but does not bind to an F(ab) present in the multispecific antigen-binding molecule. [H-2] The antibody according to [H-1], wherein the F(ab) has at least one pair of amino acid residues forming a VH-VL interface or a CH1-CL interface, and all amino acid residues forming the amino acid residue pair have the same type of charge. [H-3] An isolated antibody according to [H-1] or [H-2]. [H-4] An antibody that can bind to an F(ab) present in a light chain / heavy chain mispaired variant (mutant) of a bispecific antibody but not present in the bispecific antibody. [H-5] An antibody that can bind to an F(ab), wherein the F(ab) contains at least one pair of amino acid residues forming a VH-VL or CH1-CL interface, and all of the at least one pair of amino acid residues have the same type of charge. [H-6] The antibody according to [H-5], wherein the amino acid residues forming the VH-VL interface or the CH1-CL interface are identical. [H-7] The antibody according to [H-5] or [H-6], which does not bind to F(ab) when the pair of amino acid residues forming the VH-VL interface or the CH1-CL interface do not have the same type of charge.

[0018] Sensorgrams showing binding to standard antibodies having various heavy chain and light chain pairs when HL-PP-0-rFc is used as the analyte. Sensorgrams showing binding to standard antibodies having various heavy chain and light chain pairs when HL-NN-0-rFc is used as the analyte. Sensorgrams showing binding to standard antibodies having various heavy chain and light chain pairs when HL-PP-1-rFc is used as the analyte. Sensorgrams showing binding to standard antibodies having various heavy chain and light chain pairs when HL-PP-2-rFc is used as the analyte. Sensorgrams showing binding to standard antibodies having various heavy chain and light chain pairs when HL-PP-3-rFc is used as the analyte. Sensorgrams showing binding to standard antibodies having various heavy chain and light chain pairs when HL-PP-4-rFc is used as the analyte. Sensorgrams showing binding to standard antibodies having various heavy chain and light chain pairs when HL-PP-5-rFc is used as the analyte. Sensorgrams showing binding to standard antibodies having various heavy chain and light chain pairs when HL-PP-7-rFc is used as the analyte. Sensorgrams showing binding to standard antibodies having various heavy chain and light chain pairs when HL-NN-1-rFc is used as the analyte. Sensorgrams showing binding to standard antibodies having various heavy chain and light chain pairs when HL-NN-2-rFc is used as the analyte. Sensorgrams showing binding to standard antibodies having various heavy chain and light chain pairs when HL-NN-3-rFc is used as the analyte. Sensorgrams showing binding to standard antibodies having various heavy chain and light chain pairs when HL-NN-4-rFc is used as the analyte. Sensorgrams showing binding to standard antibodies having various heavy chain and light chain pairs when HL-PP-6-hFc is immobilized on a sensor chip. Sensorgrams showing binding to standard antibodies having various heavy chain and light chain pairs when HL-PP-8-hFc is immobilized on a sensor chip. Sensorgrams showing binding of HL-PP-9-hFc to standard antibodies having various heavy chain / light chain pairs when immobilized on a sensor chip. Sensorgrams showing binding of HL-PP-10-hFc to standard antibodies having various heavy chain / light chain pairs when immobilized on a sensor chip. Sensorgrams showing binding of HL-PP-11-hFc to standard antibodies having various heavy chain / light chain pairs when immobilized on a sensor chip. Sensorgrams showing binding of HL-PP-12-hFc to standard antibodies having various heavy chain / light chain pairs when immobilized on a sensor chip.Sensorgrams of binding to standard antibodies with various heavy and light chain pairs when HL-PP-13-hFc was immobilized on a sensor chip. A concentration-dependent calibration curve is shown for the light chain exchanger C1-MRAH(N)HERL(N) / HERH(P)MRAL(P). A concentration-dependent calibration curve is shown for the light chain exchanger C3-MRAH(N)HERL(N) / HERH(P)MRAL(P). A concentration-dependent calibration curve is shown for the light chain exchanger C19-MRAH(N)HERL(N) / HERH(P)MRAL(P). A concentration-dependent calibration curve is shown for the light chain exchanger C1-MRAH(N)HERL(N) / HERH(P)MRAL(P), which was prepared using an antibody biotinylated during expression. A concentration-dependent calibration curve is shown for the light chain exchangers Vs.Gn.C1.PP / / Gn.Vs.C1.NN. 1 shows the concentration-dependent calibration curves of the light chain exchangers Mt.Om.C1.PP / / Om.Mt.C1.NN. 2 shows the concentration-dependent calibration curves of the light chain exchangers Mt.Om.C3.PP / / Om.Mt.C3.NN.

[0019] Antigen-binding molecule As used herein, the term "antigen-binding molecule" refers, in its broadest sense, to a molecule that specifically binds to an antigenic determinant (epitope). In one embodiment, the antigen-binding molecule is an antibody, an antibody fragment, or an antibody derivative. In one embodiment, the antigen-binding molecule of the present disclosure is a multispecific antigen-binding molecule, for example, a bispecific antibody.

[0020] As used herein, the terms "specifically bind" or "specific binding" refer to binding in a state in which one of the specifically binding molecules does not show any significant binding to any molecules other than the one or more molecules to which it binds. The term also applies to cases in which an antigen-binding domain is specific for a particular epitope among multiple epitopes contained in an antigen. Furthermore, when the epitope to which the antigen-binding domain binds is contained in multiple different antigens, an antigen-binding molecule having the antigen-binding domain can bind to various antigens containing that epitope.

[0021] Antibodies The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0022] "Binding activity" refers to the strength of the total non-covalent interactions between one or more binding sites of a molecule (e.g., an antibody) and the molecule's binding partner (e.g., an antigen). In one embodiment, antigen-binding molecules and antibodies analyzed by the methods of the present disclosure can be tested for their antigen-binding activity by known methods, such as ELISA, Western blot, biolayer interferometry, surface plasmon resonance, etc.

[0023] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variants (e.g., variants containing naturally occurring mutations or variants that arise during the production of a monoclonal antibody preparation; such variants are typically present in small amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present disclosure can be produced by a variety of techniques, including, but not limited to, hybridoma technology, recombinant DNA technology, phage display technology, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.

[0024] "Native antibodies" refer to immunoglobulin molecules with various naturally occurring structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain contains a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain contains a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be assigned to one of two types, called kappa (κ) or lambda (λ).

[0025] Antigen-Binding Domain As used herein, the term "antigen-binding domain" refers, in one embodiment, to a region that specifically binds to and is complementary to a part or all of an antigen. As used herein, an antigen-binding molecule comprises an antigen-binding domain. When an antigen has a large molecular weight, the antigen-binding domain can bind only to a specific part of the antigen. This specific part is called an epitope. In one embodiment, the antigen-binding domain comprises an antibody fragment that binds to a specific antigen. The antigen-binding domain can be provided by one or more antibody "variable domains." In a non-limiting embodiment, the antigen-binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Examples of such antigen-binding domains include "scFv (single chain Fv)," "single chain antibody," "Fv," "scFv2 (single chain Fv 2)," "Fab," or "Fab'." In another embodiment, the antigen-binding domain comprises a non-antibody protein or a fragment thereof that binds to a specific antigen. In one embodiment, the antigen-binding domain includes VHH, minibody, and antibody fragments containing these. In one embodiment, the antigen binding domain comprises a hinge region.

[0026] Variable Domain The term "variable region" or "variable domain" refers to the heavy or light chain domain of an antibody that is involved in binding an antigen-binding molecule or antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies typically have similar structures, each containing four conserved framework regions (FR) and three hypervariable regions (HVR) or complementarity-determining regions (CDR). (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by screening a complementary library of VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to that antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0027] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence (the "complementarity determining region" or "CDR") and / or forms structurally defined loops (the "hypervariable loops") and / or contains antigen-contacting residues (the "antigen contacts"). Typically, antibodies contain six HVRs (CDRs): three in the VH (H1, H2, H3; H-CDR1, H-CDR2, H-CDR3) and three in the VL (L1, L2, L3; L-CDR1, L-CDR2, L-CDR3). Exemplary HVRs herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) combinations of (a), (b), and / or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0028] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0029] Constant Regions In one embodiment of the present disclosure, the constant region is preferably an antibody constant region, more preferably an IgG1, IgG2, IgG3, or IgG4 antibody constant region, and even more preferably a human IgG1, IgG2, IgG3, or IgG4 antibody constant region. In another embodiment of the present disclosure, the constant region is preferably a heavy chain constant region, more preferably an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, and even more preferably a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. The amino acid sequences of the human IgG1 constant region, human IgG2 constant region, human IgG3 constant region, and human IgG4 constant region are known. For the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, several allotype sequences due to genetic polymorphisms are described in "Sequences of proteins of immunological interest," NIH Publication No. 91-3242, and any of these may be used in the present disclosure. Note that the amino acid-altered constant regions of the present disclosure may also contain other amino acid mutations or modifications as long as they contain the amino acid mutations of the present disclosure.

[0030] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain, except that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) residues of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also known as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0031] Antibody Fragments "Antibody fragment" and "antibody fragment" refer to molecules other than intact antibodies that contain a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); single-chain Fab (scFab); single-domain antibodies; and multispecific antibodies formed from antibody fragments.

[0032] F(ab) As used herein, "F(ab)" (also referred to as Fab or Fab') is composed of a light chain consisting of a VL (light chain variable region) and a CL (light chain constant region), and a portion consisting of a VH (heavy chain variable region) and a CH1 (γ1 region of the heavy chain constant region) of the heavy chain, and may have a structure in which the portion of the heavy chain and the light chain are linked by a disulfide bond at the C-terminal region. F(ab) may also contain a portion of the hinge region. F(ab) as an antibody fragment may be simply referred to as F(ab) or an F(ab) fragment. F(ab) as a portion of an antigen-binding molecule (e.g., an antibody) is also simply referred to as F(ab), but may also be referred to as an F(ab) portion or F(ab) region to distinguish it from an F(ab) fragment. As used herein, F(ab) may or may not have antigen-binding activity. In an exemplary embodiment, both of the two F(ab)s contained in a multispecific antigen-binding molecule (H1L1 / H2L2) have antigen-binding activity. In one exemplary embodiment, the antigen-binding activity of one or both of the two F(ab)s contained in the light-chain-exchanged form (H1L2 / H2L1) of the multispecific antigen-binding molecule is weaker than the antigen-binding activity of the two F(ab)s contained in the multispecific antigen-binding molecule. In one embodiment, one or both of the two F(ab)s contained in the light-chain-exchanged form (L-chain-exchanged form) of the multispecific antigen-binding molecule have substantially no antigen-binding activity.

[0033] Multispecific antigen-binding molecules In one embodiment, the antigen-binding molecule of the present disclosure is a multispecific antigen-binding molecule (e.g., a bispecific antigen-binding molecule). In one embodiment, a multispecific antigen-binding molecule is a monoclonal antigen-binding molecule having binding specificities at at least two different sites. In one embodiment, a multispecific antigen-binding molecule may bind to two different epitopes on a single antigen. In another embodiment, a multispecific antigen-binding molecule comprises one antigen-binding site having binding specificities for two or more different antigens. For example, a multispecific antigen-binding molecule comprises a first antigen-binding site for two or more different antigens and a second antigen-binding site for one antigen. Such a multispecific antigen-binding molecule is a molecule having two different antigen-binding sites and having binding specificities for three or more different antigens. Multispecific antigen-binding molecules can be prepared as full-length antibodies or antibody fragments.

[0034] Recombinant Methods and Constructs Antigen-binding molecules can be produced using recombinant methods and constructs, for example, as described in U.S. Patent No. 4,816,567. In a non-limiting example of such production, an isolated nucleic acid encoding the antigen-binding molecule is used. Such a nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antigen-binding molecule (e.g., the light chain and / or the heavy chain of the antigen-binding molecule). One or more vectors (e.g., expression vectors) comprising such nucleic acids may be used. In an exemplary embodiment, a host cell comprising such a nucleic acid or vector is used. In one such embodiment, the host cell contains (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antigen-binding molecule and an amino acid sequence comprising the VH of the antigen-binding molecule, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antigen-binding molecule and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antigen-binding molecule. In one embodiment, the host cell is a eukaryotic cell (e.g., a Chinese hamster ovary (CHO) cell) or a lymphoid cell (e.g., a Y0, NS0, or Sp2 / 0 cell). In one embodiment, a method for producing an antigen-binding molecule is provided, comprising culturing a host cell containing a nucleic acid encoding the antigen-binding molecule as described above under conditions suitable for expression of the antigen-binding molecule, and optionally recovering the antigen-binding molecule from the host cell (or host cell culture medium).

[0035] For recombinant production of antigen-binding molecules, nucleic acids encoding the antigen-binding molecules are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antigen-binding molecules).

[0036] Suitable host cells for cloning or expressing vectors encoding antigen-binding molecules include prokaryotic or eukaryotic cells as described herein. For example, antigen-binding molecules may be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (Also see Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, antigen-binding molecules may be isolated in a soluble fraction from bacterial cell paste and further purified.

[0037] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antigen-binding molecules with partial or fully human glycosylation patterns, are suitable cloning or expression hosts for antigen-binding molecule-encoding vectors. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0038] Host cells derived from multicellular organisms (invertebrates and vertebrates) are also suitable for expressing glycosylated antigen-binding molecules. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for use in conjugation with insect cells, particularly transformation of Spodoptera frugiperda cells.

[0039] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES™ technology for producing antigen-binding molecules in transgenic plants).

[0040] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that have been adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7); human embryonic kidney (293 or 293 cells, e.g., as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney (BHK) cells; mouse Sertoli cells (TM4 cells, e.g., as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney (CV1); African green monkey kidney (VERO-76); human cervical carcinoma (HELA); canine kidney (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., as described in Mather et al., Annals NY Acad. Sci. 383:44-68). (1982)); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for producing antigen-binding molecules, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0041] Multispecific antigen-binding molecule (H1L1 / H2L2) and its light chain exchange product (H1L2 / H2L1) Herein, a first F(ab) comprising or consisting of a first heavy chain variable domain (VH contained in H1) and a first light chain variable domain (VL contained in L1) is referred to as H1L1, a second F(ab) comprising or consisting of a second heavy chain variable domain (VH contained in H2) and a second light chain variable domain (VL contained in L2) is referred to as H2L2, and a multispecific antigen-binding molecule comprising H1L1 and H2L2 is referred to as H1L1 / H2L2 (in one embodiment, a "bispecific antibody having the correct pair"). Furthermore, a third F(ab) comprising or consisting of a first heavy chain variable domain (VH contained in H1) and a second light chain variable domain (VL contained in L2) is referred to as H1L2, a fourth F(ab) comprising or consisting of a second heavy chain variable domain (VH contained in H2) and a first light chain variable domain (VL contained in L1) is referred to as H2L1, and a molecule comprising H1L2 and H2L1, i.e., a light chain exchanger of H1L1 / H2L2, is referred to as H1L2 / H2L1 (in one embodiment, it may also be referred to as a "mispair variant," "mispair mutant," "bispecific antibody having a mispair," "mispair antibody," or "mispair light chain exchanger"). In particular, in one embodiment, "mispair variant" refers to a light chain exchanger (H1L2 / H2L1). Thus, the light-chain-exchanged product herein refers to an impurity molecule (a "by-product" in one embodiment) generated by the inversion (mispairing) of the combination of two different heavy chains and two different light chains that constitute the multispecific antigen-binding molecule of interest. In one embodiment, the light-chain-exchanged product H1L2 / H2L1 can be included as a non-target molecule in the process of producing the multispecific antigen-binding molecule H1L1 / H2L2 (in one embodiment, a method using FAST-Ig technology).In one embodiment, the multispecific antigen-binding molecule H1L1 / H2L2 comprises a first F(ab) (designated H1L1) comprising or consisting of a heavy chain portion (H1) comprising or consisting of a first heavy chain variable (VH) domain and a CH1 domain, and a light chain portion (L1) comprising or consisting of a first light chain variable (VL) domain and a CL domain, and a second F(ab) (designated H2L2) comprising or consisting of a heavy chain portion (H2) comprising or consisting of a second heavy chain variable (VH) domain and a CH1 domain, and a light chain portion (L2) comprising or consisting of a second light chain variable (VL) domain and a CL domain. In one embodiment, H1L2 / H2L1, a light chain exchange product of the multispecific antigen-binding molecule H1L1 / H2L2, comprises a third F(ab) (referred to as H1L2) comprising or consisting of a heavy chain portion (H1) comprising or consisting of a first heavy chain variable (VH) domain and a CH1 domain, and a light chain portion (L2) comprising or consisting of a second light chain variable (VL) domain and a CL domain, and a fourth F(ab) (referred to as H2L1) comprising or consisting of a heavy chain portion (H2) comprising or consisting of a second heavy chain variable (VH) domain and a CH1 domain, and a light chain portion (L1) comprising or consisting of a first light chain variable (VL) domain and a CL domain. The first F(ab) H1L1 and the second F(ab) H2L2 may or may not have antigen-binding activity. In an exemplary embodiment, both H1L1 and H2L2 have antigen-binding activity. The third F(ab), H1L2, and the fourth F(ab), H2L1, may or may not have antigen-binding activity. In one embodiment, one or both of H1L2 and H2L1 have antigen-binding activity. In one embodiment, the antigen-binding activity of one or both of H1L2 and H2L1 is weaker than that of one or both of H1L1 and H2L2. In one embodiment, one or both of H1L2 and H2L1 have substantially no antigen-binding activity.In one embodiment, the light chain (L1 / L2) in the present disclosure is, for example, "Kappa / Kappa," "Kappa / Lambda," or "Lambda / Lambda."

[0042] Immunoassay In one embodiment, the method of the present disclosure is carried out using a ligand binding assay, which is an immunoassay. In one embodiment, the present disclosure relates to a method for measuring the presence and / or amount of mispair variants (light chain exchangers) in a sample. Examples of the ligand binding assay that can be used include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), surface plasmon resonance (SPR), electrochemiluminescence (ECL), and kinetic exclusion assay (KinExA®) (Drake et al., 2004, Analytical Biochemistry 328:35-43).

[0043] In one embodiment, a method can be used that has a smaller spatial size of the reaction space, a larger specific surface area of ​​the solid phase surface, and a shorter assay time than methods using microtiter plates, such as, but not limited to, a method using a microfluidic chip, a disk, or beads, more specifically, a method using, for example, the KinExA (registered trademark) or Gyrolab (registered trademark) immunoassay system (Fraley et al., 2013, Bioanalysis 5: 1765-74). In one embodiment, the present disclosure relates to a method for quantifying or detecting mispair variants in a sample using a Single Molecule Array method, an Alpha (Amplified Luminescence Proximity Homogeneous Assay) method, or a time-resolved fluorescence method.

[0044] In one embodiment, the method of the present disclosure is a method for detecting mispair variants in a sample, the method comprising the steps of: (1) contacting a first binder (in one embodiment, a first detection antibody) with a sample containing mispair variants and allowing the first binder to bind to the mispair variant; (2) allowing the mispair variant bound to the first binder to bind to a second binder (in one embodiment, a second detection antibody); and (3) detecting the second binder bound to the mispair variant. In one embodiment, the binder of the present disclosure is an antibody (detection antibody). In one embodiment, the first binder and the second binder are the first detection antibody and the second detection antibody, respectively.

[0045] In one aspect, the method of the present disclosure relates to a method for quantifying mispair variants in a sample, the method comprising the steps of: (1) contacting a first binder with a sample containing mispair variants to allow the first binder to bind to the mispair variant; (2) allowing the mispair variant bound to the first binder to bind to a second binder; and (3) quantifying the second binder bound to the mispair variant (measuring the amount of binding of the second binder to the mispair variant).

[0046] In one embodiment, the first conjugate of the present disclosure is bound to a solid phase.

[0047] In one aspect, the method of the present disclosure relates to a method for quantifying or detecting mispair variants in a sample, the method comprising the steps of: (1) contacting a first binder bound to a solid phase with a sample containing mispair variants to bind the mispair variants to the first binder; (2) binding the mispair variants bound to the first binder bound to the solid phase with a second binder; and (3) quantifying or detecting the second binder bound to the mispair variants.

[0048] In one aspect, the method of the present disclosure relates to a method for quantifying or detecting mispair variants in a sample, the method comprising the steps of: (1)(a) contacting a sample containing mispair variants with a first binder to bind the mispair variants to the first binder; (1)(b) binding the first binder bound to the mispair variant to a solid phase; (2) binding the mispair variants bound to the first binder bound to the solid phase to a second binder; and (3) quantifying or detecting the second binder bound to the mispair variant.

[0049] In one aspect, the method of the present disclosure is a method for quantifying or detecting mispair variants in a sample, the method comprising the steps of: (1) + (2) (a) contacting a first binder and a second binder with a sample containing mispair variants, thereby allowing the first binder and the second binder to bind to the mispair variant, respectively; (b) binding the first binder bound to the mispair variant bound to the second binder to a solid phase; and (3) detecting the second binder bound to the mispair variant. Here, in the step (1) + (2) (a), the first binder and the second binder may be simultaneously contacted with the sample containing the mispair variant. Alternatively, the first binder may be contacted with the sample containing the mispair variant before the second binder is contacted with the mispair variant. Alternatively, the second binder may be contacted with the sample containing the mispair variant before the first binder is contacted with the mispair variant.

[0050] In one embodiment, the method of the present disclosure may include one or more washing steps.

[0051] The first binder may bind to the mispair variant, and may be selected from, for example, a polypeptide, an antibody, an antibody fragment, a fusion polypeptide comprising an antibody or antibody fragment and a non-antibody polypeptide, a fusion polypeptide comprising an antibody or antibody fragment and a soluble receptor, or a fusion polypeptide comprising an antibody or antibody fragment and a peptidic binding molecule.

[0052] The second binder may be selected from, for example, a polypeptide, an antibody, a fusion polypeptide comprising an antibody or antibody fragment and a non-antibody polypeptide, a fusion polypeptide comprising an antibody or antibody fragment and a soluble receptor, or a fusion polypeptide comprising an antibody or antibody fragment and a peptidic binding molecule. In one embodiment, the second binder binds to the mispair variant without inhibiting the binding between the first binder and the mispair variant.

[0053] In one embodiment, the first binder and the second binder each bind to a different portion constituting the mispair variant. In one embodiment, when the mispair variant comprises a first F(ab) and a second F(ab), the first binder binds to the first F(ab) and the second binder binds to the second F(ab).

[0054] In one embodiment, the method of the present disclosure is performed without labeling or immobilizing the mispair variant to be detected. In one embodiment, the method of the present disclosure is performed by labeling or immobilizing the first or second conjugate (in one embodiment, the first or second detection antibody). Labeling refers to, but is not limited to, modification with a luminescent label, a chemiluminescent label, an electrochemiluminescent label, a fluorescent label, digoxigenin, biotin, avidin, or a radioactive label. Immobilization refers to, but is not limited to, binding or fixing, directly or indirectly, to a solid phase such as a bead, a disk, a microfluidic chip, a magnetic particle, or a microtiter plate. In one embodiment, the conjugate (e.g., the detection antibody) of the present disclosure is biotinylated or fluorescently labeled.

[0055] In one embodiment, the conjugate of the present disclosure is labeled. The label may be, but is not limited to, a luminescent label, a chemiluminescent label, an electrochemiluminescent label, a fluorescent label, digoxigenin, biotin, or a radioactive label. In one embodiment, the conjugate of the present disclosure (e.g., a detection antibody) is fluorescently labeled.

[0056] The solid phase as used herein may be a bead, a disk, a microfluidic chip, a magnetic particle, or a microtiter plate, and is not limited thereto as long as it can achieve the objectives of the present disclosure.

[0057] Methods well known to those skilled in the art can be used to bind the conjugate of the present disclosure to a solid phase. In one embodiment, when the conjugate of the present disclosure is a polypeptide, the conjugate of the present disclosure is bound to the solid phase by chemical bonding via the N-terminal group and / or ε-amino group (lysine) of the amino acid backbone of the polypeptide, the ε-amino group, carboxy functional group, sulfhydryl functional group, hydroxyl functional group, and / or phenol functional group of a different lysine, and / or a sugar alcohol group of the carbohydrate structure of the polypeptide.

[0058] In one embodiment, the conjugates of the present disclosure are bound to a solid phase by passive adsorption, as described, for example, by Butler, JE, in "Solid Phases in Immunoassay" (1996) 205-225 and Diamandis, EP, and Christopoulos, TK (Editors), in "Immunoassay" (1996) Academic Press (San Diego).

[0059] In one embodiment, the conjugate of the present disclosure is bound to a solid phase via a specific binding pair. In one embodiment, such a binding pair (first component / second component) is selected from streptavidin or avidin / biotin, antibody / antigen (see, e.g., Hermanson, GT, et al., Bioconjugate Techniques, Academic Press (1996)), lectin / polysaccharide, steroid / steroid-binding protein, hormone / hormone receptor, enzyme / substrate, IgG / protein A and / or protein G, etc. In one embodiment, the first conjugate is linked to biotin, and binding occurs via avidin or streptavidin immobilized on the solid phase. In one embodiment, the conjugate of the present disclosure (e.g., detection antibody) is biotinylated.

[0060] As used herein, "detection" includes quantitative and qualitative detection, and examples of qualitative detection include simply measuring whether a mispair variant is present, measuring whether a certain amount of a mispair variant is present, and comparing the amount of a mispair variant with that of another sample (e.g., a control sample). On the other hand, examples of quantitative detection include measuring the concentration of a mispair variant and measuring the amount of a mispair variant.

[0061] Method for Quantifying or Detecting Mispaired Variants of Multispecific Antigen-Binding Molecules In one embodiment, the present disclosure relates to a method for quantifying or detecting mispaired variants of multispecific antigen-binding molecules (sometimes referred to as the "method of the present disclosure"). In one embodiment, the multispecific antigen-binding molecule of the present disclosure is a bispecific antibody. Bispecific antibodies with non-common L chains have two types of H chains (H1, H2) and two types of L chains (L1, L2). Therefore, in addition to the target antibody (H1L1-H2L2), nine impurities resulting from mismatching of H chains and L chains are generated during the production process. Up to eight of these impurities can be suppressed or separated using knobs-into-holes or charge control technology, which suppress mispairing between heavy chains, or Art-Ig technology, which creates a pI difference between each chain. However, the remaining type (H1L2-H2L1, hereafter referred to as the L-chain exchanger) shares physical properties, such as theoretical pI, with the target antibody, making separation during the manufacturing process difficult and potentially resulting in a certain amount of L-chain exchanger being present in the composition as an impurity. In this case, the identical physical properties make it difficult to separate and identify the L-chain exchanger in the composition, and a method for detecting L-chain exchangers is needed from a quality control perspective. In designing bispecific antibodies with non-common L chains, to promote the association of the target antibody (H1L1-H2L2), the correct pair of H and L chains is substituted with positively and negatively charged (or negatively and positively charged) amino acid residues (using FAST-Ig technology). While the target antibody contains substitutions of amino acid residues with opposite charges in the paired H and L chains, the L-chain exchanger (H1L2-H2L1) contains substitutions of amino acid residues with the same charge in the H and L chains. In other words, in an L-chain exchanged antibody, if the light chain-heavy chain interface of one arm has a positive-positive substitution, the light chain-heavy chain interface of the other arm will have a negative-negative substitution. The present inventors have found that by using an antibody that recognizes the light chain-heavy chain interface with a positive-positive substitution (hereinafter sometimes referred to as an "anti-PP antibody") and an antibody that recognizes the light chain-heavy chain interface with a negative-negative substitution (hereinafter sometimes referred to as an "anti-NN antibody"), it is possible to detect L-chain exchanged antibodies generated as mispairs of bispecific antibodies with non-common L chains.One embodiment of the method of the present disclosure relates to a method for quantifying or detecting mispair variants of a multispecific antigen-binding molecule using an anti-PP antibody or an anti-NN antibody. In one embodiment, the mispair variants of the multispecific antigen-binding molecule of the present disclosure comprise at least two different F(ab)s that are not present in a multispecific antigen-binding molecule with a correct pair (of H chain and L chain). A "mispair" may be referred to as, for example, a "mismatch" or an "incorrect pair," etc. A variant may be referred to as, for example, a "mutant," an "altered form," etc. A "correct pair" may be referred to as, for example, a "correct match" or a "normal pair," etc. In one embodiment of the method of the present disclosure, the mispair variants are a method for quantifying or detecting mispair variants using an antibody that binds to the mispair but does not bind to the correct pair. In one embodiment, the mispair variants of the present disclosure comprise an F(ab) having at least one first pair of amino acid residues that constitute the VH-VL interface or the CH1-CL interface, and both amino acid residues that constitute the first pair of amino acid residues are positively or negatively charged. In one embodiment, a mispaired variant of a multispecific antigen-binding molecule of the present disclosure comprises a first and a second F(ab), wherein the first F(ab) has at least a first pair of amino acid residues forming a VH-VL interface or a CH1-CL interface, and both amino acid residues forming the first pair of amino acid residues are positively charged; and the second F(ab) has at least a second pair of amino acid residues forming a VH-VL interface or a CH1-CL interface, and both amino acid residues forming the second pair of amino acid residues are negatively charged.

[0062] In one embodiment, the method of the present disclosure is a method comprising contacting a sample containing mispair variants to be detected with a detection antibody. In one embodiment, the method of the present disclosure is a method for quantifying or detecting mispair variants of a multispecific antigen-binding molecule, wherein the mispair variants comprise at least two different F(ab)s that are not present in the multispecific antigen-binding molecule, and the method comprises the steps of: (a) contacting a first detection antibody with the sample containing the mispair variants; (b) contacting a second detection antibody with the mispair variants bound to the first detection antibody; and (c) quantifying or detecting the second detection antibody bound to the mispair variants, wherein one of the first or second detection antibodies binds to one of the F(ab), and the other binds to the other F(ab). In one embodiment, "step" may be expressed as, for example, "step" or "stage."

[0063] In one embodiment, a bispecific antibody having a correct pair of heavy and light chains comprises heavy chain A, heavy chain B, light chain A, and light chain B, and includes heavy chain A associated with light chain A (direct pairing) and heavy chain B associated with light chain B (direct pairing). In one embodiment, a mispaired antibody (mispair variant) comprises heavy chain A, heavy chain B, light chain A, and light chain B, and includes heavy chain A associated with light chain B (first mispaired heavy chain A-light chain B) and heavy chain B associated with light chain A (second mispaired heavy chain B-light chain A). In one embodiment, the heavy chain A and light chain A each contain amino acid residues that promote the association of heavy chain A and light chain A. In one embodiment, the heavy chain B and light chain B each contain amino acid residues that promote the association of heavy chain B and light chain B.

[0064] In one embodiment, the detection antibody of the present disclosure includes a first and a second detection antibody, one of which binds to one F(ab) of the mispaired variant of the present disclosure, and the other of which binds to the other F(ab) of the mispaired variant of the present disclosure. In one embodiment, the detection antibody of the present disclosure is such that one of the first and second detection antibodies is capable of binding to an assembly of heavy chain A and light chain B of the mispaired antibody (mispaired variant) of the present disclosure (first mispaired heavy chain A-light chain B), and the other of the first and second detection antibody is capable of binding to an assembly of heavy chain B and light chain A of the mispaired antibody (mispaired variant) of the present disclosure (second mispaired heavy chain B-light chain A).

[0065] In one embodiment, the method of the present disclosure relates to a method for detecting or quantifying a mispaired antibody in a mixture of a bispecific antibody and its mispaired by-product, wherein the bispecific antibody comprises heavy chain A, heavy chain B, light chain A, and light chain B, and comprises heavy chain A associated with light chain A (correct pairing, normal pairing) and heavy chain B associated with light chain B (correct pairing, normal pairing), and the mispaired antibody comprises heavy chain A, heavy chain B, light chain A, and light chain B, and comprises heavy chain A associated with light chain B (first mispaired heavy chain A-light chain B) and heavy chain B associated with light chain A (second mispaired heavy chain B-light chain A), and the method includes any of the following: (a) contacting the mixture of the bispecific antibody and its mispaired by-product with a first detection antibody; (b) contacting the antibody bound to the first detection antibody of step (a) with a second detection antibody; (c) detecting the amount of binding of the second detection antibody to the antibody bound to the first detection antibody in step (b) (sometimes referred to as "quantitative detection of binding," "quantitation of binding," or "measurement of binding amount"); in one embodiment, one of the first and second detection antibodies is capable of binding to an aggregate of heavy chain A and light chain B of the mispaired antibody (first mispaired heavy chain A-light chain B), and the other of the first and second detection antibodies is capable of binding to an aggregate of heavy chain B and light chain A of the mispaired antibody (second mispaired heavy chain B-light chain A).

[0066] In one embodiment, the first or second detection antibody exhibits strong binding to the association of heavy chain A and light chain B (the first mispaired heavy chain A-light chain B) and / or the association of heavy chain A and light chain B (the second mispaired heavy chain A-light chain B). In one embodiment, the first or second detection antibody exhibits stronger binding to the association of heavy chain A and light chain B (the first mispaired heavy chain A-light chain B) and / or the association of heavy chain B and light chain A (the second mispaired heavy chain B-light chain A) than to the association of the correct pair of heavy chain A and light chain A and / or the correct pair of heavy chain B and light chain B. In one embodiment, the first and / or second detection antibody does not substantially bind to the association of the correct pair of heavy chain A and light chain A and / or the correct pair of heavy chain B and light chain B. In one embodiment, the detection antibody of the present disclosure binds to the F(ab) present in a mispaired variant of a multispecific antigen-binding molecule but does not bind to the F(ab) present in the multispecific antigen-binding molecule. In one embodiment of the present disclosure, "does not bind" means "does not substantially bind." In one embodiment, "does not substantially bind" refers to a binding activity that is 80% or less, 50% or less, 30% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of the binding activity to the F(ab) present in the mispaired variant of the multispecific antigen-binding molecule. Examples of the F(ab) present in the mispaired variant of a multispecific antigen-binding molecule include the F(ab) used as an immunogen in the production method of the present disclosure.

[0067] In one embodiment, the method of the present disclosure relates to a method for detecting or quantifying mispaired by-product antibodies in a mixture of a bispecific antibody and its mispaired by-product antibodies, wherein the bispecific antibody comprises heavy chain A, heavy chain B, light chain A, and light chain B in the bispecific antibody, and comprises heavy chain A associated with light chain A (first correct pair: heavy chain A-light chain A) and heavy chain B associated with light chain B (second correct pair: heavy chain B-light chain B), and wherein the mispaired by-product antibodies (e.g., mispaired variants) comprise an association of heavy chain A and light chain B (first mispaired heavy chain A-light chain B) and / or an association of heavy chain B and light chain A (second mispaired heavy chain B-light chain A), the method comprising: (a) contacting the mixture of the bispecific antibody and its mispaired by-products (e.g., mispaired variants) with a detection antibody, (b) contacting the detection antibody, which is capable of binding to the heavy chain A and light chain B aggregate (first mispaired heavy chain A-light chain B) or the heavy chain B and light chain A aggregate (second mispaired heavy chain B-light chain A), and which does not substantially bind to the heavy chain A and light chain A aggregate (first correct paired heavy chain A-light chain A) or the heavy chain B and light chain B aggregate (second correct paired heavy chain B-light chain B); and (b) detecting the amount of binding of the detection antibody to the mispaired by-product antibody of (a).

[0068] In one embodiment, the method of the present disclosure relates to a method that satisfies any of the following: (a) contacting a mixture of the bispecific antibody and its mispaired by-product with a first detection antibody; (b) contacting the antibody bound to the first detection antibody in step (a) with a second detection antibody; and (c) detecting the amount of binding of the second detection antibody to the antibody bound to the first detection antibody in step (b), wherein one of the first and second detection antibodies is capable of binding to an aggregate of heavy chain A and light chain B of the related mispaired antibody (first mispaired heavy chain A-light chain B), and the other of the first and second detection antibodies is capable of binding to an aggregate of heavy chain B and light chain A of the related mispaired antibody (second mispaired heavy chain B-light chain A). In one embodiment of the method of the present disclosure, the mispaired variant comprises two F(ab)s that are not present in the correctly paired bispecific antibody, and the two F(ab)s are different from each other.

[0069] In one embodiment, the method of the present disclosure comprises the following steps: (a) contacting a sample (or solution) containing a mispaired variant with a first detection antibody; (b) contacting the mispaired variant bound by the first detection antibody in step (a) with a second detection antibody; (c) detecting the second detection antibody (or detecting the second detection antibody bound to the mispaired variant bound by the first detection antibody in step (b)); wherein one of the first detection antibody and the second detection antibody is capable of binding to one of the two F(ab)s contained in the mispaired variant, and the other of the first detection antibody and the second detection antibody is capable of binding to the other of the two F(ab)s contained in the mispaired variant. In one embodiment, the method of the present disclosure relates to a method for quantifying / detecting mispair variants of a bispecific antibody, wherein the mispair variants comprise the following F(ab): a first F(ab) comprising at least one pair of amino acid residues that form a VH-VL interface or a CH1-CL interface, and wherein the at least one pair of amino acid residues both have a positive charge; and a second F(ab) comprising at least one pair of amino acid residues that form a VH-VL interface or a CH1-CL interface, and wherein the at least one pair of amino acid residues both have a negative charge.

[0070] In one embodiment, the mispaired variant of the present disclosure comprises a first and a second F(ab), wherein the first F(ab) has at least a first pair of amino acid residues forming a VH-VL interface or a CH1-CL interface, and both amino acid residues forming the first pair of amino acid residues are positively charged; the second F(ab) has at least a second pair of amino acid residues forming a VH-VL interface or a CH1-CL interface, and both amino acid residues forming the second pair of amino acid residues are negatively charged; and one of the first or second detection antibodies binds to one of the first or second F(ab), and the other binds to the other of the first or second F(ab).

[0071] In one embodiment, the mispair variants of the present disclosure include: (1) a first and a second F(ab) in which the amino acids at positions 147 and 175 in CH1 and the amino acids at positions 131 and 160 in CL are charge-controlled to repel each other; (2) a first and a second F(ab) in which the amino acids at positions 147 and 175 in CH1 and the amino acids at positions 131 and 180 in CL are charge-controlled to repel each other; or (3) a first and a second F(ab) in which the amino acids at positions 147 and 175 in CH1 and the amino acids at positions 131, 160, and 180 in CL are charge-controlled to repel each other; wherein the position numbers of CH1 are according to EU numbering and the position numbers of CL are according to Kabat numbering.

[0072] In one embodiment, the method of the present disclosure relates to a method in which the mispair variant F(ab) comprises the following amino acid residues: (1) Ks at positions 147 and 175 in CH1; and Ks at positions 131 and 160 in CL; (2) Ks at positions 147 and 175 in CH1; and Ks at positions 131 and 180 in CL; (3) Ks at positions 147 and 175 in CH1; and Ks at positions 160 and 180 in CL; (4) Ks at positions 147 and 213 in CH1; and Ks at positions 123 and 131 in CL; (5) Ks at positions 147 and 213 in CH1; and Ks at positions 123 and 160 in CL; (6) Ks at positions 147 and 213 in CH1; and K at positions 123 and 180 in CL; (7) K at positions 175 and 213 in CH1; and K at positions 123 and 131 in CL; (8) K at positions 175 and 213 in CH1; and K at positions 123 and 160 in CL; (9) K at positions 175 and 213 in CH1; and K at positions 123 and 180 in CL; (10) K at positions 147 and 175 in CH1; and K at positions 131, 160, and 180 in CL; (11) K at positions 147, 175, and 213 in CH1; and K at positions 123, 131, 160, and 180 in CL; Here, the position numbers of CH1 follow EU numbering, and the position numbers of CL follow Kabat numbering.

[0073] In one embodiment, the method of the present disclosure relates to a method in which the mispair variant F(ab) comprises the following amino acid residues: (1) E at positions 147 and 175 in CH1; and E at positions 131 and 160 in CL; (2) E at positions 147 and 175 in CH1; and E at positions 131 and 180 in CL; (3) E at positions 147 and 175 in CH1; and E at positions 160 and 180 in CL; (4) E at positions 147 and 213 in CH1; and E at positions 123 and 131 in CL; (5) E at positions 147 and 213 in CH1; and E at positions 123 and 160 in CL; (6) E at positions 147 and 213 in CH1; and E at positions 123 and 180 in CL; (7) E at positions 175 and 213 in CH1; and E at positions 123 and 131 in CL; (8) E at positions 175 and 213 in CH1; and E at positions 123 and 160 in CL; (9) E at positions 175 and 213 in CH1; and E at positions 123 and 180 in CL; (10) E at positions 147 and 175 in CH1; and E at positions 131, 160, and 180 in CL; (11) E at positions 147, 175, and 213 in CH1; and E at positions 123, 131, 160, and 180 in CL; Here, the position numbers of CH1 follow EU numbering, and the position numbers of CL follow Kabat numbering.

[0074] One embodiment of the method of the present disclosure relates to a method for detecting L-chain exchangers using an anti-NN antibody and / or an anti-PP antibody. Another embodiment of the method of the present disclosure relates to a method for detecting mispaired variants using an antibody that binds to the mispair but does not bind to the correct pair. One embodiment of the method of the present disclosure relates to a method for detecting mispaired variants using an antibody that binds to the mispair of antibodies produced by FAST-Ig technology but does not bind to the correct pair of antibodies produced by FAST-Ig technology.

[0075] FAST-Ig Technology In one embodiment, the mispaired variant of the present disclosure is generated as a by-product antibody of an antibody produced by the FAST-Ig technology. In one embodiment, the mispaired variant of the present disclosure is a light chain (L chain)-exchanged antibody of an antibody produced by the FAST-Ig technology. In designing a bispecific antibody with non-common L chains, the H chain and L chain that constitute the correct pair are substituted with amino acid residues that have a positive charge and a negative charge (or a negative charge and a positive charge), respectively (using the FAST-Ig technology) to promote the association of the target antibody (H1L1-H2L2). In one embodiment, the FAST-Ig technology relates to an antibody in which the association of the heavy chain and light chain is controlled, and in which the heavy chain and light chain that constitute the antibody assemble in the desired heavy chain and light chain combination, the amino acid residues at predetermined positions in the heavy chain constant region (CH1) and the light chain constant region are mutually attracting (in one embodiment, "not repelling") in terms of charge. By using amino acid residues that attract each other electrically at specific positions in the heavy chain constant region (CH1) and light chain constant region of the desired heavy chain and light chain combination, the desired heavy chain and light chain combination can be formed, for example, by utilizing the attractive force of charges.

[0076] In the present disclosure, the term "interface" generally refers to the surface at which association (interaction) occurs, and the amino acid residues forming the interface generally refer to one or more amino acid residues contained in the polypeptide region involved in the association, more preferably amino acid residues that come close to each other during association and are involved in the interaction. Specific examples of such interactions include those formed by hydrogen bonds, electrostatic interactions, salt bridges, etc., between amino acid residues that come close to each other during association.

[0077] In the present disclosure, "amino acid residues forming an interface" refers, more specifically, to amino acid residues contained in a polypeptide region that constitutes the interface. For example, a polypeptide region that constitutes the interface refers to a polypeptide region that is responsible for selective intermolecular association (interaction) in an antibody, ligand, receptor, substrate, etc. Specific examples of such a polypeptide region in an antibody include a heavy chain constant region, heavy chain variable region, light chain constant region, light chain variable region, etc.

[0078] In the present disclosure, "modification" of an amino acid residue specifically refers to substituting an original amino acid residue with another amino acid residue, deleting an original amino acid residue, adding a new amino acid residue, etc., but preferably refers to substituting an original amino acid residue with another amino acid residue.

[0079] In one embodiment of the FAST-Ig technology, antibodies are produced in which amino acid residues involved in association at the polypeptide interface have amino acid residues that attract each other electrically. By modifying the amino acid residues involved in association at the polypeptide interface in the antibody to amino acid residues that attract each other electrically, it is thought that the association between these amino acid residues is promoted, for example, by the attractive force of their charges.

[0080] In one embodiment of the FAST-Ig technology, the amino acid residues that are modified are those that are close to each other during association between polypeptide regions that form an interface.

[0081] In one embodiment of the FAST-Ig technology, amino acid residues that are close to each other during association can be found, for example, by analyzing the three-dimensional structure of the polypeptide and examining the amino acid sequence of the polypeptide region that forms an interface during association of the polypeptide. In one embodiment of the FAST-Ig technology, amino acid residues that are close to each other at the interface are targets for "modification" of the antibody.

[0082] Some amino acids are known to be electrically charged. Lysine (K), arginine (R), and histidine (H) are generally known as positively charged amino acids. Negatively charged amino acids include aspartic acid (D) and glutamic acid (E). Uncharged or nonpolar amino acids include alanine (A), asparagine (N), cysteine ​​(C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V). Therefore, in one aspect of the FAST-Ig technology, amino acids that are mutually charge-repulsive (have the same type of charge) mean: (1) one amino acid is a positively charged amino acid and the other amino acid is also a positively charged amino acid; or (2) one amino acid is a negatively charged amino acid and the other amino acid is also a negatively charged amino acid.

[0083] In one embodiment of the FAST-Ig technology, amino acids that do not repel each other in terms of charge mean: (1) one amino acid is positively charged and the other amino acid is negatively charged, (2) one amino acid is positively charged and the other amino acid is uncharged or a nonpolar amino acid, (3) one amino acid is negatively charged and the other amino acid is uncharged or a nonpolar amino acid, or (4) neither amino acid is charged or a nonpolar amino acid. In another embodiment of the FAST-Ig technology, amino acids that attract each other in terms of charge mean one amino acid is positively charged and the other amino acid is negatively charged.

[0084] In one embodiment, the FAST-Ig technology can be applied to an IgG-type bispecific antibody having two heavy chain constant regions CH1 (CH1-A and CH1-B) and two light chain constant regions (CL-A and CL-B).

[0085] When it is desired to use the FAST-Ig technology to obtain a bispecific antibody that recognizes one epitope via an antigen-binding region comprising a first CH1-A and a first CL-A, and another epitope via an antigen-binding region comprising a second CH1-B and a second CL-B, if each of the four chains is expressed in the production of the antibody, theoretically, 10 types of antibody molecules can be produced.

[0086] In this case, for example, by controlling the association between CH1-A and CL-B and / or between CH1-B and CL-A to be inhibited, it is possible to preferentially obtain the desired antibody molecule.

[0087] For example, amino acid residues forming the interface between CH1-A and CL-B can be modified to positively charged amino acid residues, and amino acid residues forming the interface between CH1-B and CL-A can be modified to negatively charged amino acid residues. This modification inhibits unintended association between CH1-A and CL-B because both amino acid residues forming the interface are positively charged, inhibits association between CH1-B and CL-A because both amino acid residues forming the interface are negatively charged, and inhibits unintended association between CH1-A and CL-B and between CH1-B and CL-A because the amino acid residues forming the interface have the same charge. As a result, antibodies exhibiting the desired association between CH1-A and CL-A and the desired association between CH1-B and CL-B can be efficiently obtained. One embodiment of the method disclosed herein relates to a method for quantifying or detecting molecules that form associations between CH1-A and CL-B and between CH1-B and CL-A, which are generated as by-products of antibodies produced by FAST-Ig technology. One embodiment of the method of the present disclosure relates to a method for quantifying or detecting mispair variants using an antibody that binds to the mispair of antibodies generated by the FAST-Ig technology but does not bind to the correct pair generated by the FAST-Ig technology.

[0088] In one embodiment of the FAST-Ig technology, specific examples of amino acid residues that are close (facing or in contact) at the interface between CH1 and CL when they associate include the following combinations: - Lysine (K) at EU numbering position 147 in CH1 and the threonine (T) or serine (S) at EU numbering position 180 in the CL that faces (contacts) it. - Lysine (K) at EU numbering position 147 in CH1 and the serine (S) or threonine (T) at EU numbering position 131 in the CL that faces (contacts) it. - Glutamine (Q) at EU numbering position 175 in CH1 and the serine (S) or threonine (T) at EU numbering position 131 in the CL that faces (contacts) it. - Glutamine (Q) at EU numbering position 175 in CH1 and the glutamine (Q) or threonine (T) at EU numbering position 160 in the CL that faces (contacts) it. - Lysine (K) at EU numbering position 213 in CH1 and the glutamic acid (E) at EU numbering position 123 in the CL that faces (contacts) it.

[0089] In one embodiment of the FAST-Ig technology, specific examples of combinations of amino acid residues that are mutually attractive in terms of charge include the following combinations: - The amino acid residue at position 175 (EU numbering) in CH1 is lysine (K), and the amino acid residues at positions 180, 131, and 160 (EU numbering) in CL are all glutamic acid (E). - The amino acid residues at positions 147 and 175 (EU numbering) in CH1 are glutamic acid (E), and the amino acid residues at positions 180, 131, and 160 (EU numbering) in CL are all lysine (K). In another embodiment of the FAST-Ig technology, further examples of combinations of amino acid residues that are mutually attractive in terms of charge include the amino acid residue at position 213 (EU numbering) in CH1 being glutamic acid (E), and the amino acid residue at position 123 (EU numbering) in CL being lysine (K).

[0090] In one embodiment, bispecific antibodies (mispaired variants; L-chain exchanged products) generated as a by-product of bispecific antibodies produced by the FAST-Ig technology are charge-repulsive at the association between CH1-A and CL-B and between CH1-B and CL-A. In one embodiment, the "mutually charge-repulsive amino acid residues" are selected from amino acid residues included in either of the following groups (X) or (Y): (X) glutamic acid (E) or aspartic acid (D), (Y) lysine (K), arginine (R), or histidine (H).

[0091] In one embodiment of the FAST-Ig technology, amino acid residues that repel or attract each other electrically are close to each other when associated. In one embodiment, similar to the interface between the CH1 and CL regions, sites corresponding to amino acid residues that repel or attract each other electrically can be found for the interface between the desired heavy and light chain variable regions by homology modeling using commercially available software, and the amino acid residues at these sites can be modified as appropriate. Modifications of heavy and light chains using the FAST-Ig technology are sometimes referred to herein as "FAST-Ig modification."

[0092] Detection Antibodies One embodiment of the detection antibody of the present disclosure is an antibody that binds to F(ab) present in a mispaired variant of a multispecific antigen-binding molecule but does not bind to F(ab) present in a multispecific antigen-binding molecule with the correct pair. In one embodiment, "binding" or "capable of binding" to F(ab) can be expressed as substantially binding to F(ab), specifically binding to F(ab), recognizing F(ab), or having binding activity to F(ab). In one embodiment, "not binding" or "not capable of binding" to F(ab) can be expressed as not substantially binding to F(ab), not recognizing F(ab), or not having binding activity to F(ab). One embodiment of the detection antibody of the present disclosure is the anti-PP antibody or anti-NN antibody described above. In one embodiment, the detection antibody of the present disclosure is an antibody that binds to a mispaired bispecific antibody generated, for example, by FAST-Ig technology. In one embodiment, the detection antibody of the present disclosure is an antibody that recognizes a mispaired heavy chain and light chain containing FAST-Ig modifications in the CH1 / CL region. In one embodiment, the detection antibody of the present disclosure binds to an F(ab) in which both amino acid residues of at least one pair of amino acid residues constituting the VH-VL interface or the CH1-CL interface have the same charge. In one embodiment, the detection antibody of the present disclosure binds to an F(ab) of an L-chain-exchanged product of a bispecific antibody generated by the FAST-Ig technology (a mispaired F(ab)), and in another embodiment, does not bind to an F(ab) of a correct pair of a bispecific antibody generated by the FAST-Ig technology.

[0093] In one embodiment, the detection antibody of the present disclosure binds to an F(ab) having a charge-repulsive VH-VL interface or CH1-CL interface. In one embodiment, the detection antibody of the present disclosure binds to an F(ab) comprising a charge-modified polypeptide. In one embodiment, the detection antibody of the present disclosure binds to an F(ab) having a charge-controlled VH-VL interface or CH1-CL interface. In one embodiment, "charge-modified" means that the amino acid residues that form desired pairs at the VH-VL interface or CH1-CL interface are modified so that they have opposite charges (they attract each other) and / or the amino acid residues that form undesired pairs have the same charges (they repel each other). In one embodiment of the detection antibody of the present disclosure, the antibody binds to the following F(ab): (1) an F(ab) in which the charge is controlled so that the amino acids at positions 147 and 175 in CH1 and the amino acids at positions 131 and 160 in CL repel each other; (2) an F(ab) in which the charge is controlled so that the amino acids at positions 147 and 175 in CH1 and the amino acids at positions 131 and 180 in CL repel each other; or (3) an F(ab) in which the charge is controlled so that the amino acids at positions 147 and 175 in CH1 and the amino acids at positions 131, 160, and 180 in CL repel each other;

[0094] In one embodiment, the detection antibody of the present disclosure binds to an F(ab) comprising the following amino acid residues present in a mispair variant of the present disclosure: (1) Ks at positions 147 and 175 in CH1; and Ks at positions 131 and 160 in CL; (2) Ks at positions 147 and 175 in CH1; and Ks at positions 131 and 180 in CL; (3) Ks at positions 147 and 175 in CH1; and Ks at positions 160 and 180 in CL; (4) Ks at positions 147 and 213 in CH1; and Ks at positions 123 and 131 in CL; (5) Ks at positions 147 and 213 in CH1; and Ks at positions 123 and 160 in CL; (6) Ks at positions 147 and 213 in CH1; and K at positions 123 and 180 in CL; (7) K at positions 175 and 213 in CH1; and K at positions 123 and 131 in CL; (8) K at positions 175 and 213 in CH1; and K at positions 123 and 160 in CL; (9) K at positions 175 and 213 in CH1; and K at positions 123 and 180 in CL; (10) K at positions 147 and 175 in CH1; and K at positions 131, 160, and 180 in CL; or (11) K at positions 147, 175, and 213 in CH1; and K at positions 123, 131, 160, and 180 in CL.

[0095] Examples of embodiments of the detection antibody of the present disclosure include the following: (1) an antibody that can bind to an F(ab) in which positions 147 and 175 in CH1 are K and positions 131 and 160 in CL are K, but an F(ab) in which positions 147 and 175 in CH1 are K and positions 131 and 160 in CL are E; and / or an antibody that cannot bind to an F(ab) in which positions 147 and 174 in CH1 are E and positions 131 and 160 in CL; (2) an antibody that can bind to an F(ab) in which positions 147 and 175 in CH1 are K and positions 131 and 180 in CL, but an F(ab) in which positions 147 and 175 in CH1 are K and positions 131 and 180 in CL are E; and / or an antibody that cannot bind to an F(ab) in which positions 147 and 175 in CH1 are E and positions 131 and 180 in CL are K; (3) an antibody that can bind to an F(ab) in which positions 147 and 175 in CH1 are K and positions 160 and 180 in CL are K, but an F(ab) in which positions 147 and 175 in CH1 are K and positions 160 and 180 in CL are E; and / or an antibody that cannot bind to an F(ab) in which positions 147 and 175 in CH1 are E and positions 160 and 180 in CL; (4) an antibody that can bind to an F(ab) in which positions 147 and 213 in CH1 are K and positions 123 and 131 in CL, but (5) An antibody that can bind to an F(ab) in which positions 147 and 213 in CH1 are K and positions 123 and 131 in CL are K; and / or an antibody that cannot bind to an F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 131 in CL are K; (6) An antibody that can bind to an F(ab) in which positions 147 and 213 in CH1 are K and positions 123 and 160 in CL are K; and / or an antibody that cannot bind to an F(ab) in which positions 147 and 213 in CH1 are K and positions 123 and 160 in CL;(6) An antibody capable of binding to an F(ab) in which positions 147 and 213 in CH1 are K and positions 123 and 180 in CL are K, but an F(ab) in which positions 147 and 213 in CH1 are K and positions 123 and 180 in CL are E; and / or an antibody incapable of binding to an F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 180 in CL are K; (7) An antibody capable of binding to an F(ab) in which positions 175 and 213 in CH1 are K and positions 123 and 131 in CL are K, but an F(ab) in which positions 175 and 213 in CH1 are K and positions 123 and 131 in CL are E; and / or an antibody that cannot bind to an F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 131 in CL are K; (8) an antibody that can bind to an F(ab) in which positions 175 and 213 in CH1 are K and positions 123 and 160 in CL are K, but an F(ab) in which positions 175 and 213 in CH1 are K and positions 123 and 131 in CL are E; and / or an antibody that cannot bind to an F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 131 in CL; (9) an antibody that can bind to an F(ab) in which positions 175 and 213 in CH1 are K and positions 123 and 180 in CL, but (10) An antibody that can bind to an F(ab) in which positions 147 and 175 in CH1 are K and positions 131, 160, and 180 in CL are K; and / or an antibody that cannot bind to an F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 180 in CL are K; (11) An antibody that can bind to an F(ab) in which positions 147 and 175 in CH1 are K and positions 131, 160, and 180 in CL are K; and / or an antibody that cannot bind to an F(ab) in which positions 147 and 175 in CH1 are E and positions 131, 160, and 180 in CL;or (11) an antibody capable of binding to an F(ab) in which positions 147, 175, and 213 in CH1 are K and positions 123, 131, 160, and 180 in CL are K, but an F(ab) in which positions 147, 175, and 213 in CH1 are K and positions 123, 131, 160, and 180 in CL are E; and / or an antibody incapable of binding to an F(ab) in which positions 147, 175, and 213 in CH1 are E and positions 123, 131, 160, and 180 in CL are K;

[0096] In one embodiment, the detection antibody of the present disclosure binds to an F(ab) comprising the following amino acid residues present in a mispaired variant of the present disclosure: (1) E at positions 147 and 175 in CH1; and E at positions 131 and 160 in CL; (2) E at positions 147 and 175 in CH1; and E at positions 131 and 180 in CL; (3) E at positions 147 and 175 in CH1; and E at positions 160 and 180 in CL; (4) E at positions 147 and 213 in CH1; and E at positions 123 and 131 in CL; (5) E at positions 147 and 213 in CH1; and E at positions 123 and 160 in CL; (6) E at positions 147 and 213 in CH1; and E at positions 123 and 180 in CL; (7) E at positions 175 and 213 in CH1; and E at positions 123 and 131 in CL; (8) E at positions 175 and 213 in CH1; and E at positions 123 and 160 in CL; (9) E at positions 175 and 213 in CH1; and E at positions 123 and 180 in CL; (10) E at positions 147 and 175 in CH1; and E at positions 131, 160, and 180 in CL; or (11) E at positions 147, 175, and 213 in CH1; and E at positions 123, 131, 160, and 180 in CL.

[0097] Examples of detection antibodies according to the present disclosure include the following: (1) an antibody capable of binding to an F(ab) having E at positions 147 and 175 in CH1 and E at positions 131 and 160 in CL, but not to an F(ab) having E at positions 147 and 175 in CH1 and E at positions 131 and 160 in CL; and / or an F(ab) having E at positions 147 and 174 in CH1 and K at positions 131 and 160 in CL; (2) an antibody capable of binding to an F(ab) having E at positions 147 and 175 in CH1 and E at positions 131 and 180 in CL, but not to an F(ab) having K at positions 147 and 175 in CH1 and E at positions 131 and 180 in CL; and / or an antibody that cannot bind to an F(ab) in which positions 147 and 175 in CH1 are E and positions 131 and 180 in CL are K; (3) an antibody that can bind to an F(ab) in which positions 147 and 175 in CH1 are E and positions 160 and 180 in CL, but an F(ab) in which positions 147 and 175 in CH1 are K and positions 160 and 180 in CL; and / or an antibody that cannot bind to an F(ab) in which positions 147 and 175 in CH1 are E and positions 160 and 180 in CL; (4) an antibody that can bind to an F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 131 in CL, but (5) An antibody that can bind to an F(ab) in which positions 147 and 213 in CH1 are K and positions 123 and 131 in CL are E; and / or an antibody that cannot bind to an F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 131 in CL are K; (6) An antibody that can bind to an F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 160 in CL, but can bind to an F(ab) in which positions 147 and 213 in CH1 are K and positions 123 and 160 in CL; and / or an antibody that cannot bind to an F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 160 in CL;(6) An antibody capable of binding to F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 180 in CL are E, but an F(ab) in which positions 147 and 213 in CH1 are K and positions 123 and 180 in CL are E; and / or an antibody incapable of binding to F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 180 in CL are K; (7) An antibody capable of binding to F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 131 in CL, but an F(ab) in which positions 175 and 213 in CH1 are K and positions 123 and 131 in CL are E; and / or an antibody that cannot bind to an F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 131 in CL are K; (8) an antibody that can bind to an F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 160 in CL, but an F(ab) in which positions 175 and 213 in CH1 are K and positions 123 and 131 in CL; and / or an antibody that cannot bind to an F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 131 in CL; (9) an antibody that can bind to an F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 180 in CL, but (10) An antibody that can bind to an F(ab) in which positions 147 and 175 in CH1 are E and positions 131, 160, and 180 in CL are E; and / or an antibody that cannot bind to an F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 180 in CL are K; (11) An antibody that can bind to an F(ab) in which positions 147 and 175 in CH1 are E and positions 131, 160, and 180 in CL are E; but an F(ab) in which positions 147 and 175 in CH1 are K and positions 131, 160, and 180 in CL are E; and / or an antibody that cannot bind to an F(ab) in which positions 147 and 175 in CH1 are E and positions 131, 160, and 180 in CL are K;or (11) an antibody capable of binding to an F(ab) in which positions 147, 175, and 213 in CH1 are E and positions 123, 131, 160, and 180 in CL are E, but an F(ab) in which positions 147, 175, and 213 in CH1 are K and positions 123, 131, 160, and 180 in CL; and / or an antibody incapable of binding to an F(ab) in which positions 147, 175, and 213 in CH1 are E and positions 123, 131, 160, and 180 in CL are K;

[0098] Examples of one embodiment of the detection antibody of the present disclosure include antibodies comprising the following VH and VL: (1) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 64, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 69, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 75; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 80, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 90; (2) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 70, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 76; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 81, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 91; (3) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 70, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 76; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 86, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 91; (4) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 70, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 76; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 92; (5) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 70, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 77;and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 92; (6) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 70, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 77; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 86, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 92; (7) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 70, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 77; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 93; (8) VH comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 70, and HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 77; and VL comprising LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 81, LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 92; (9) VH comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 66, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 71, and HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 77; and VL comprising LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 87, and LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 94; (10) VH comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 66, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 71, and HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 77;and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 93; (11) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 72, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 78; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 93; (12) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 71, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 78; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 93; (13) VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 66, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 71, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 78; and VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 93; (14) VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 66, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 73, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 77; and VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 93; (15) VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 67, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 74, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 79;and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 83, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 88, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 95; (16) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 67, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 74, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 79; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 84, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 88, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 95; (17) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 67, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 74, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 79; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 83, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 89, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 95; (18) (19) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 68, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 74, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 79; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 84, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 88, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 95; or (20) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 68, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 74, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 79; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 83, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 89, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 96.

[0099] An example of a detection antibody of the present disclosure is an antibody comprising the following VH and VL: (1) VH comprising the amino acid sequence set forth in SEQ ID NO: 8 and VL comprising the amino acid sequence set forth in SEQ ID NO: 31; (2) VH comprising the amino acid sequence set forth in SEQ ID NO: 8 and VL comprising the amino acid sequence set forth in SEQ ID NO: 32; (3) VH comprising the amino acid sequence set forth in SEQ ID NO: 8 and VL comprising the amino acid sequence set forth in SEQ ID NO: 33; (4) VH comprising the amino acid sequence set forth in SEQ ID NO: 9 and VL comprising the amino acid sequence set forth in SEQ ID NO: 33; (5) VH comprising the amino acid sequence set forth in SEQ ID NO: 9 and VL comprising the amino acid sequence set forth in SEQ ID NO: 34; (6) VH comprising the amino acid sequence set forth in SEQ ID NO: 9 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (7) VH comprising the amino acid sequence set forth in SEQ ID NO: 9 and VL comprising the amino acid sequence set forth in SEQ ID NO: 36; (8) VH comprising the amino acid sequence set forth in SEQ ID NO: 10 and VL comprising the amino acid sequence set forth in SEQ ID NO: 37; (9) VH comprising the amino acid sequence set forth in SEQ ID NO: 10 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (10) VH comprising the amino acid sequence set forth in SEQ ID NO: 11 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (11) VH comprising the amino acid sequence set forth in SEQ ID NO: 12 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (12) VH comprising the amino acid sequence set forth in SEQ ID NO: 13 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (13) VH comprising the amino acid sequence set forth in SEQ ID NO: 14 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (14) VH comprising the amino acid sequence set forth in SEQ ID NO: 9 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (15) VH comprising the amino acid sequence set forth in SEQ ID NO: 10 and VL comprising the amino acid sequence set forth in SEQ ID NO: 37; (16) VH comprising the amino acid sequence set forth in SEQ ID NO: 10 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (17) VH comprising the amino acid sequence set forth in SEQ ID NO: 11 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35;(18) VH comprising the amino acid sequence set forth in SEQ ID NO: 12 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (19) VH comprising the amino acid sequence set forth in SEQ ID NO: 13 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (20) VH comprising the amino acid sequence set forth in SEQ ID NO: 14 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (21) VH comprising the amino acid sequence set forth in SEQ ID NO: 9 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (22) VH comprising the amino acid sequence set forth in SEQ ID NO: 10 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (23) VH comprising the amino acid sequence set forth in SEQ ID NO: 11 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (24) VH comprising the amino acid sequence set forth in SEQ ID NO: 12 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (25) VH comprising the amino acid sequence set forth in SEQ ID NO: 13 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (26) (27) a VH comprising the amino acid sequence set forth in SEQ ID NO: 14 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (28) a VH comprising the amino acid sequence set forth in SEQ ID NO: 7 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 39; (29) a VH comprising the amino acid sequence set forth in SEQ ID NO: 15 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 38; (30) a VH comprising the amino acid sequence set forth in SEQ ID NO: 15 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 40; (31) a VH comprising the amino acid sequence set forth in SEQ ID NO: 7 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 30; (32) a VH comprising the amino acid sequence set forth in SEQ ID NO: 6 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 29; or (33) a VH comprising the amino acid sequence set forth in SEQ ID NO: 7 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 30. In one embodiment, the antibody of the present disclosure is an isolated (separated) antibody.

[0100] Nucleic Acids / Vectors The present disclosure relates to nucleic acids encoding the antibodies or antigen-binding fragments thereof of the present disclosure. In one aspect, the present disclosure relates to vectors carrying the nucleic acids.

[0101] The nucleic acid of the present disclosure is typically carried (inserted) into an appropriate vector and introduced into a host cell. The vector is not particularly limited as long as it stably retains the inserted nucleic acid. For example, if Escherichia coli is used as the host, cloning vectors such as the pBluescript vector (Stratagene) can be used. Various commercially available vectors can also be used. When using a vector for the purpose of producing the polypeptide of the present disclosure, an expression vector is particularly useful. Expression vectors are not particularly limited as long as they express a polypeptide in a test tube, in Escherichia coli, in cultured cells, or in an individual organism. Examples of such vectors include the pBEST vector (Promega) for in vitro expression, the pET vector (Invitrogen) for Escherichia coli, the pME18S-FL3 vector (GenBank Accession No. AB009864) for cultured cells, and the pME18S vector (Mol Cell Biol. 8:466-472 (1988)) for individual organisms. The DNA of the present disclosure can be inserted into a vector using, for example, the In-Fusion Advantage PCR Cloning Kit (Clontech).

[0102] Host Cells The present disclosure also relates to host cells carrying the above-described nucleic acids. The host cells are not particularly limited, and various host cells, such as Escherichia coli and various animal cells, can be used depending on the purpose. The host cells can be used, for example, as production systems for producing or expressing the antibodies or polypeptides of the present disclosure. Production systems for producing polypeptides include in vitro and in vivo production systems. In vitro production systems include production systems using eukaryotic cells and production systems using prokaryotic cells.

[0103] Eukaryotic cells that can be used as host cells include, for example, animal cells, plant cells, and fungal cells. Examples of animal cells include mammalian cells such as CHO (J. Exp. Med. (1995) 108: 945), COS, 3T3, myeloma, BHK (baby hamster kidney), HeLa, C127, HEK293, Bowes melanoma cells, and Vero; amphibian cells such as Xenopus oocytes (Valle et al., Nature (1981) 291: 338-340); and insect cells such as Drosophila S2, Sf9, Sf21, and Tn5. For the expression of antibodies of the present disclosure, CHO-DG44, CHO-DX11B, COS7, and BHK cells are preferably used. For the purpose of large-scale expression, CHO cells are used as an example of animal cells. Vectors can be introduced into host cells by known methods, such as the calcium phosphate method, the DEAE-dextran method, the cationic liposome DOTAP (Boehringer Mannheim), electroporation (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Sections 9.1-9.9), lipofection, the lipofectamine method (GIBCO-BRL), and microinjection. Alternatively, the FreeStyle 293 Expression System (Invitrogen) can be used to carry out processes from gene introduction to polypeptide expression.

[0104] For example, cells derived from Nicotiana tabacum are known as a protein production system for plant cells, and the antibodies of the present disclosure can be produced by callus culture of these cells. Protein expression systems using fungal cells, such as yeast cells of the genus Saccharomyces (e.g., Saccharomyces cerevisiae, Saccharomyces pombe), and filamentous fungi cells of the genus Aspergillus (e.g., Aspergillus niger), are known and can be used as hosts for producing the antibodies of the present disclosure.

[0105] When prokaryotic cells are used, there are production systems using bacterial cells, such as Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, and Bacillus subtilis, in addition to the aforementioned E. coli, which can be used to produce the antibodies of the present disclosure.

[0106] Antibody Production When producing antibodies using the host cells of the present disclosure, host cells transformed with an expression vector containing a polynucleotide encoding the antibody of the present disclosure are cultured to express the polynucleotide. Culturing can be carried out according to known methods. For example, when animal cells are used as hosts, culture media such as DMEM, MEM, RPMI1640, and IMDM can be used. In this case, serum supplements such as FBS and fetal calf serum (FCS) can be used in combination, or the cells can be cultured in a serum-free culture. The pH during culture is, for example, approximately 6 to 8. Culture is typically carried out at approximately 30 to 40°C for approximately 15 to 200 hours, with medium replacement, aeration, and agitation as necessary.

[0107] On the other hand, systems for producing polypeptides in vivo include, for example, production systems using animals and production systems using plants. A target polynucleotide is introduced into these animals or plants, and the polypeptide is produced in the animal or plant body and then recovered. In the present disclosure, the term "host" includes these animals and plants.

[0108] To allow secretion of a polypeptide expressed in a host cell into the lumen of the endoplasmic reticulum, into the periplasmic space, or into the extracellular environment, appropriate secretion signals can be incorporated into the polypeptide of interest. These signals can be endogenous to the polypeptide of interest or heterologous signals.

[0109] In the above production method, when the polypeptide of the present disclosure is secreted into the medium, the medium is collected, whereas when the polypeptide of the present disclosure is produced intracellularly, the cells are first lysed and then the polypeptide is collected.

[0110] When animals are used, there are production systems using mammals and insects. Mammals that can be used include goats, pigs, sheep, mice, and cows (Vicki Glaser, SPECTRUM Biotechnology Applications (1993)). When mammals are used, transgenic animals can also be used.

[0111] For example, a polynucleotide encoding an antibody of the present disclosure is prepared as a fusion gene with a gene encoding a polypeptide specifically produced in milk, such as caprine beta-casein. A polynucleotide fragment containing this fusion gene is then injected into a goat embryo, and the embryo is then implanted into a female goat. The antibody of interest can be obtained from the milk produced by the transgenic goat born to the goat that received the embryo, or its offspring. To increase the amount of milk containing the antibody produced by the transgenic goat, appropriate hormones may be administered to the transgenic goat (Ebert et al., Bio / Technology (1994) 12: 699-702).

[0112] Furthermore, as an insect for producing the antibody of the present disclosure, for example, silkworms can be used. When using silkworms, the silkworms can be infected with a baculovirus into which a polynucleotide encoding the antibody of interest has been inserted, and the antibody of interest can be obtained from the body fluids of the silkworms (Susumu et al., Nature (1985) 315: 592-4).

[0113] Furthermore, when using plants to produce antibodies of the present disclosure, tobacco can be used, for example. When using tobacco, a polynucleotide encoding the antibody of interest is inserted into a plant expression vector, such as pMON 530, and this vector is then introduced into bacteria such as Agrobacterium tumefaciens. This bacterium is then used to infect tobacco, such as Nicotiana tabacum, and the desired antibody can be obtained from the tobacco leaves (Ma et al., Eur. J. Immunol. (1994) 24: 131-8).

[0114] The antibody thus obtained can be isolated from inside or outside the host cell (culture medium, milk, etc.) and purified as a substantially pure, homogeneous antibody. Antibody isolation and purification can be performed using any isolation and purification method commonly used for purifying polypeptides, and is not limited in any way. For example, antibodies can be isolated and purified by an appropriate combination of methods such as ammonium sulfate or ethanol precipitation, acid extraction, chromatography columns, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization.

[0115] Examples of chromatography include affinity chromatography, ion exchange chromatography such as anion exchange chromatography or cation exchange chromatography, phosphocellulose chromatography, hydrophobic (interaction) chromatography, gel filtration, reversed-phase chromatography, adsorption chromatography, hydroxylapatite chromatography, and lectin chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed. Daniel R. Marshak et al. (1996) Cold Spring Harbor Laboratory Press). These chromatographies can be performed using liquid-phase chromatography such as HPLC and FPLC. Examples of columns used in affinity chromatography include Protein A columns and Protein G columns. Examples of columns using Protein A include Hyper D, POROS, and Sepharose FF (Pharmacia).

[0116] If necessary, before or after antibody purification, an appropriate protein-modifying enzyme such as trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, or glucosidase can be used to optionally modify or partially remove peptides.

[0117] Production Method In one embodiment, the present disclosure relates to a method for producing an antibody of the present disclosure, comprising the steps of culturing a host cell of the present disclosure as described above and recovering the antibody from the cell culture. In one embodiment, the present disclosure relates to a method for producing an antibody for detecting a mispaired variant of a multispecific antigen-binding molecule, comprising the step of immunizing with an F(ab) present in the mispaired variant. In this production method, in one embodiment, the F(ab) has at least one pair of amino acid residues that form the VH-VL interface or the CH1-CL interface, and both amino acid residues that form the amino acid residue pair have the same charge. In one embodiment, the F(ab) that can be used as an immunogen is an F(ab) that contains FAST-Ig modification in the VH / VL region or the CH1 / CL region.

[0118] In one embodiment, the immunogenic F(ab) may be: (1) a Fab in which charge is controlled so that the amino acids at positions 147 and 175 in CH1 repel each other, and the amino acids at positions 131 and 160 in CL repel each other; (2) a Fab in which charge is controlled so that the amino acids at positions 147 and 175 in CH1 repel each other, and the amino acids at positions 131 and 180 in CL repel each other; or (3) a Fab in which charge is controlled so that the amino acids at positions 147 and 175 in CH1 repel each other, and the amino acids at positions 131, 160, and 180 in CL repel each other. Here, the position numbers for CH1 are based on EU numbering, and the position numbers for CL are based on Kabat numbering.

[0119] In one embodiment, the immunogenic F(ab) includes F(ab) containing the following amino acid residues present in the mispair variant: (1) K at positions 147 and 175 in CH1; and K at positions 131 and 160 in CL; (2) K at positions 147 and 175 in CH1; and K at positions 131 and 180 in CL; (3) K at positions 147 and 175 in CH1; and K at positions 160 and 180 in CL; (4) K at positions 147 and 213 in CH1; and K at positions 123 and 131 in CL; (5) K at positions 147 and 213 in CH1; and K at positions 123 and 160 in CL; (6) K at positions 147 and 213 in CH1; (7) K at positions 175 and 213 in CH1; and K at positions 123 and 131 in CL; (8) K at positions 175 and 213 in CH1; and K at positions 123 and 160 in CL; (9) K at positions 175 and 213 in CH1; and K at positions 123 and 180 in CL; (10) K at positions 147 and 175 in CH1; and K at positions 131, 160, and 180 in CL; or (11) K at positions 147, 175, and 213 in CH1; and K at positions 123, 131, 160, and 180 in CL.

[0120] In one embodiment, the immunogenic F(ab) includes F(ab) containing the following amino acid residues present in mispaired variants: (1) E at positions 147 and 175 in CH1; and E at positions 131 and 160 in CL; (2) E at positions 147 and 175 in CH1; and E at positions 131 and 180 in CL; (3) E at positions 147 and 175 in CH1; and E at positions 160 and 180 in CL; (4) E at positions 147 and 213 in CH1; and E at positions 123 and 131 in CL; (5) E at positions 147 and 213 in CH1; and E at positions 123 and 160 in CL; (6) E at positions 147 and 213 in CH1; and E at positions 123 and 180 in CL; (7) E at positions 175 and 213 in CH1; and E at positions 123 and 131 in CL; (8) E at positions 175 and 213 in CH1; and E at positions 123 and 160 in CL; (9) E at positions 175 and 213 in CH1; and E at positions 123 and 180 in CL; (10) E at positions 147 and 175 in CH1; and E at positions 131, 160, and 180 in CL; or (11) E at positions 147, 175, and 213 in CH1; and E at positions 123, 131, 160, and 180 in CL.

[0121] In one embodiment, the immunogen used in the method for producing a detection antibody of the present disclosure can be the F(ab) of a mispair variant of the present disclosure (in a particular embodiment, the antibody for use as an immunogen described in the Examples section of the present specification). In one embodiment, the production method of the present disclosure comprises the step of selecting an antibody that does not bind to the F(ab) of the correct pair of multispecific antigen-binding molecules (H1L1 / H2L2). In one embodiment, the production method of the present disclosure comprises the step of selecting an antibody that specifically binds to the F(ab) present in the mispair variant of the multispecific antigen-binding molecule. In one embodiment, the method comprises the step of selecting an antibody that specifically binds to the F(ab) used as the immunogen. In one embodiment, some steps of the production method of the present disclosure can be performed using known methods.

[0122] In one embodiment, the detection antibody of the present disclosure can be produced using the above-mentioned immunogen (antigen) by a method known to those skilled in the art. For example, the antibody can be obtained from an antibody library, or can be produced by cloning a gene encoding the antibody from a hybridoma that produces a monoclonal antibody.

[0123] Many antibody libraries are already known, and methods for constructing antibody libraries are also known, so that those skilled in the art can obtain an appropriate antibody library. For example, with regard to antibody phage libraries, reference can be made to literature such as Clackson et al., Nature 1991, 352:624-8, Marks et al., J. Mol. Biol. 1991, 222:581-97, Waterhouses et al., Nucleic Acids Res. 1993, 21:2265-6, Griffiths et al., EMBO J. 1994, 13:3245-60, Vaughan et al., Nature Biotechnology 1996, 14:309-14, and Published Japanese Translation of PCT International Publication No. 1998-504970. Other known methods, such as a method using eukaryotic cells as a library (WO95 / 15393 pamphlet) or ribosome display, can also be used. Furthermore, a technique for obtaining human antibodies by panning using a human antibody library is also known. For example, the variable regions of human antibodies can be expressed on the surface of phages as single-chain fragments (scFvs) using phage display, and phages that bind to antigens can be selected. By analyzing the genes of the selected phages, the DNA sequences encoding the variable regions of human antibodies that bind to antigens can be determined.

[0124] Methods for obtaining genes encoding antibodies from hybridomas basically use known techniques, such as immunizing the hybridomas using the immunogens described above according to conventional immunization methods, fusing the resulting immune cells with known parent cells using conventional cell fusion methods, screening for monoclonal antibody-producing cells (hybridomas) using conventional screening methods, synthesizing cDNA for the variable region (V region) of the antibody from the mRNA of the resulting hybridomas using reverse transcriptase, and ligating this to DNA encoding the constant region of the desired antibody.

[0125] Antibody-producing cells can be obtained by immunizing an animal with the above-mentioned immunogen. Alternatively, lymphocytes capable of producing antibodies can be immunized in vitro to produce antibody-producing cells. Various mammals can be used as animals to be immunized, but rodents, lagomorphs, and primates are commonly used. Specific examples include rodents such as mice, rats, and hamsters; lagomorphs such as rabbits; and primates such as cynomolgus monkeys, rhesus monkeys, hamadryas baboons, and chimpanzees.

[0126] Animals can be immunized, for example, by appropriately diluting and suspending the immunogen in phosphate-buffered saline (PBS) or physiological saline, emulsifying it with an adjuvant if necessary, and then injecting it intraperitoneally or subcutaneously into the animal. Thereafter, the immunogen, preferably mixed with Freund's incomplete adjuvant, is administered several times, for example, every 4 to 21 days. Antibody production can be confirmed by measuring the titer of the desired antibody in the animal's serum using a conventional method.

[0127] Hybridomas can be produced by fusing antibody-producing cells obtained from lymphocytes or animals immunized with a desired immunogen with myeloma cells using a conventional fusing agent (e.g., polyethylene glycol) (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986, pp. 59-103). Hybridoma cells are then cultured and grown as needed, and the binding specificity, affinity, or activity of the antibodies produced by the hybridomas is measured by known analytical methods such as immunoprecipitation, radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA). Hybridomas producing antibodies with the desired binding specificity, affinity, or activity can then be subcloned, as needed, by limiting dilution or other techniques.

[0128] Subsequently, a gene encoding the antibody of interest can be cloned from a hybridoma or antibody-producing cells (such as sensitized lymphocytes) using a probe capable of specifically binding to the antibody (e.g., an oligonucleotide complementary to a sequence encoding the antibody constant region). Cloning from mRNA is also possible by RT-PCR. Immunoglobulins are classified into five different classes: IgA, IgD, IgE, IgG, and IgM. These classes are further divided into several subclasses (isotypes) (e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2, etc.). The heavy and light chains used in the production of antibodies in the present invention may be derived from antibodies belonging to any of these classes and subclasses, and are not particularly limited, although IgG is particularly preferred.

[0129] Kit In one embodiment, the present disclosure relates to a kit comprising a detection antibody. In one embodiment, the kit of the present disclosure is a kit for use in a method for quantifying or detecting mispaired variants of a multispecific antigen-binding molecule. In one embodiment, the kit may include reagents for performing an immunoassay. In one embodiment, the kit may also include reagents for labeling or immobilization. In one embodiment, the kit may include an instruction manual containing instructions for carrying out the method of the present disclosure.

[0130] All prior art documents cited in this specification are hereby incorporated by reference.

[0131] Example 1: Preparation of Immunogen Antibodies and Screening Antibodies To obtain antibodies that specifically recognize mispairing between heavy and light chains, antibodies with mispaired heavy and light chains containing FAST-Ig modifications in the CH1 / CL region were prepared as immunogens. As representative antibodies, Fab (C19-PP) was prepared from the IgG1 kappa antibody tocilizumab, in which amino acid modifications Q175K were introduced into the heavy chain and S131K, Q160K, and T180K were introduced into the light chain. Fab (C19-NN) was also prepared, in which amino acid modifications K147E and Q175E were introduced into the heavy chain and S131E, Q160E, and T180E were introduced into the light chain. These antibodies were used as immunogens. Additionally, C19-PN (heavy chain Q175K, light chain S131E, Q160E, T180E) and C19-NP (heavy chain K147E, Q175E, light chain S131K, Q160K, T180K) were prepared as antibodies with correctly paired heavy and light chains and used in the sorting screening described in Example 2. For the sorting screening, antibodies with mispaired heavy and light chains containing FAST-Ig modifications (anti-GPC3 antibodies) with variable regions different from those of tocilizumab were also used. Table 1 lists the name of each antibody, the corresponding sequence numbers of the heavy and light chains, and the amino acid modifications at the heavy and light chain interface. The position of each amino acid alteration is indicated using EU numbering for the heavy chain constant region and Kabat numbering for the heavy chain variable region, light chain variable region, and light chain constant region. The antibodies were transiently expressed in HEK293 cells using plasmid DNA encoding them and purified from the culture supernatant. Fab fragments used as immunogens were purified from antibodies digested with endoproteinase Lys-C.

[0132]

[0133] Example 2: Obtaining anti-C19-PP and anti-C19-NN antibodies Example 2.1: Generation of anti-C19-PP antibodies Anti-C19-PP antibodies were prepared, selected, and assayed as follows. Three NZW rabbits were intradermally immunized with C19-PP prepared in Example 1 (50-100 μg / dose / animal). Four repeated doses were administered over a 6-week period, followed by collection of blood and spleens. B cells that bound to C19-PP but not to C19-PN or C19-NP were isolated using a BD FACSAria. TM III Cell Sorter (BD Bioscience), and then plated and cultured according to the procedure described in WO2016098356A1. After culture, the B cell culture supernatant was collected for further analysis, and the B cell pellet was cryopreserved.

[0134] To select antibodies, binding specificity was assessed by ELISA using B cell culture supernatant. Antibodies that specifically bind to MFA-PP#2' (heavy chain Q175K, light chain S131K, Q160K, T180K) but do not bind to C19-PN (heavy chain Q175K, light chain S131E, Q160E, T180E), C19-NP (heavy chain K147E, Q175E, light chain S131K, Q160K, T180K), or MFB-NN#2' (heavy chain K147E, Q175E, light chain S131E, Q160E, T180E) were selected. The selected B cell culture supernatants were used to evaluate the binding properties of the following variants: MFA-PP#4 (heavy chain Q175K, light chain Q160K, T180K), MFA-PP#6 (heavy chain Q175K, light chain S131K, Q160K), and MFA-PP#9 (heavy chain Q175K, light chain S131K, T180K). B cells showing good cross-reactivity were suitable and selected for gene cloning.

[0135] Example 2.2: Generation of anti-C19-NN antibodies Anti-C19-NN antibodies were prepared, selected, and assayed as follows. Three NZW rabbits were immunized intradermally with C19-NN prepared in Example 1 (50-100 μg / dose / animal). Four repeated doses were administered over a six-week period, followed by collection of blood and spleens. B cells that bound to C19-NN but not to C19-PN or C19-NP were isolated using a BD FACSAria. TM III Cell Sorter (BD Bioscience), and then plated and cultured according to the procedure described in WO2016098356A1. After culture, the B cell culture supernatant was collected for further analysis, and the B cell pellet was cryopreserved.

[0136] To select antibodies, binding specificity was evaluated by ELISA using B cell culture supernatant. Antibodies that specifically bind to MFB-NN#2' (heavy chain: K147E, Q175E, light chain: S131E, Q160E, T180E) but do not bind to C19-PN (heavy chain: Q175K, light chain: S131E, Q160E, T180E), C19-NP (heavy chain: K147E, Q175E, light chain: S131K, Q160K, T180K), or MFA-PP#2' (heavy chain: Q175K, light chain: S131K, Q160K, T180K) were selected. The selected B cell culture supernatants were used to evaluate the binding properties of the following variants: MFB-NN#4 (K147E, Q175E in the heavy chain, Q160E, T180E in the light chain), MFB-NN#6 (K147E, Q175E in the heavy chain, S131E, Q160E in the light chain), and MFB-NN#9 (K147E, Q175E in the heavy chain, S131E, T180E in the light chain). B cells showing good cross-reactivity were suitable and selected for gene cloning.

[0137] Example 2.3: Gene Cloning and Preparation of Anti-C19-PP and Anti-C19-NN Antibodies. RNA from clones with the desired binding specificity was extracted from cryopreserved cell pellets using the ZR-96 Quick-RNA Kit (ZYMO RESEARCH, Catalog No. R1053). DNA encoding the antibody heavy chain variable region of the antibody produced by the selected clone was obtained and amplified by reverse transcription PCR. The DNA encoding the antibody heavy chain was then cloned by combining it with DNA encoding the rabbit IgG heavy chain constant region. DNA encoding the antibody light chain variable region was also obtained and amplified by reverse transcription PCR. The DNA encoding the antibody light chain was then cloned by combining it with DNA encoding the rabbit kappa light chain constant region. The cloned antibodies were expressed using HEK293 cells and purified from the culture supernatant. The purified antibodies were subjected to the same binding assays as those used in the selection process described above to confirm reproducibility of binding. Based on their binding properties, one clone each of anti-C19-PP antibody and anti-C19-NN antibody was selected and subjected to further analysis as described below. The sequence numbers of the VH and VL of these antibodies are shown in Table 2.

[0138]

[0139] Example 3: Evaluation of binding activity of anti-C19-PP antibody and anti-C19-NN antibody First, the binding activity of the obtained anti-C19-PP antibody and anti-C19-NN antibody was measured by bio-layer interferometry (BLI) using Octet Systems, Octet RED38 (Sartorius), or Octet HTX (Sartorius).

[0140] Example 3.1: Preparation of antibodies for antigen use containing C1, C3, and C19 modifications In addition to the C19 modification used in the immunogen, the pair of C1 and C2 modifications (collectively referred to herein as C1 modifications) and the pair of C3 and C4 modifications (collectively referred to herein as C3 modifications) described in Non-Patent Document 4 can also be used to control the association between heavy and light chains by FAST-Ig modifications. Therefore, antigen-specific antibodies containing the C1, C3, and C19 modifications (C1: heavy chain K147(E) / Q175K(E), light chain S131E(K) / Q160E(K); C3: heavy chain K147(E) / Q175K(E), light chain S131E(K) / T180E(K); C19: heavy chain K147(E) / Q175K(E), light chain S131E(K) / Q160E(K) / T180E(K); the amino acid residues listed to the right of each position number indicate one of the two Fab fragments constituting the antibody outside the parentheses and the other within the parentheses) were prepared and used for binding evaluation. The antigen-specific antibodies were generated using the anti-human RSV (respiratory syncytial virus) antibody motavizumab (IgG1, kappa) as a template. An IgG4 kappa antibody in which the heavy chain constant region was modified to that of an IgG4 type was also used. The name of each antigen antibody, the combination of amino acid modifications, and the sequence number are shown in Table 3-1. Table 3-2 also shows antibodies (HL-PP-0-rFc and HL-NN-0-rFc) in which the heavy chain CH2 and CH3 sequences of the anti-C19-PP and anti-C19-NN antibodies shown in Table 2 were modified to rabbit sequences. The HL-PP and HL-NN portions of the sample names in Table 3-2 indicate that the FAST-Ig modifications at the heavy chain-light chain interface recognize positive-positive mispairs and negative-negative mispairs, respectively. In the sample names in Tables 3-1 and 3-2, rFc and hFc indicate rabbit Fc and human Fc sequences, respectively, and the preceding descriptions (e.g., HL-PP-0 and HL-NN-0) indicate the Fab sequences of the antibodies.In the following text, the designations HL-PP-0 and HL-NN-0 refer to the Fab sequences of the antibodies. Plasmids encoding the antibodies listed in Tables 3-1 and 3-2 were constructed by methods known to those skilled in the art. Each antibody was transiently expressed in mammalian cells using the constructed plasmids by methods known to those skilled in the art, and purified by methods known to those skilled in the art.

[0141]

[0142]

[0143] Example 3.2: Measurement of binding activity using Octet Systems. The mispair-recognizing antibodies (HL-PP, HL-NN) in Tables 3-2 and 4-1 or the antigen-specific antibodies (each containing hFc) in Table 3-1 were diluted to a concentration of 10 μg / mL with TBS-T (137 mmol / L NaCl, 2.68 mmol / L KCl, 25 mmol / L Tris, pH 7.4, 0.05 w / v% Tween 20) and loaded onto Octet® AHQ Biosensors for 60 seconds. After a baseline was taken with TBS-T for 30 seconds, the antigen-specific antibodies or mispair-recognizing antibodies (HL-PP, HL-NN) (each containing rFc) were allowed to bind for 120 seconds, followed by dissociation in TBS-T buffer for 180 seconds.

[0144] All measurements were performed at 30°C. The sensor chip was regenerated three times, alternating between 10 mM glycine-HCl, pH 1.5 (Cytiva; Cat# BR-1003-54) and TBS-T buffer, as needed. Measurement data were analyzed using Octet Data Analysis HT Software Version 12, Octet BLI Analysis 12.2, or Octet Analysis Studio 12.2. The exported binding amount (nm) at each time point was divided by the antibody loading amount (nm) to calculate the binding / capture value. The time points (seconds) and binding / capture values ​​(represented as binding AU in the figures) were plotted using Spotfire® (TIBCO® Version 7.11.1.0.13) and are shown in Figures 1-1 to 1-12 and 2-1 to 2-7. In Figure 1, a mispair-recognizing antibody (containing rFc) was used as the analyte, and measurements were performed in an experimental system in which an antigen-use antibody (containing hFc) was immobilized on a Biosensor. In Figure 2, an antigen-use antibody (containing rFc) was used as the analyte, and measurements were performed in an experimental system in which a mispair-recognizing antibody (containing hFc) was immobilized on a Biosensor.

[0145] The results in Figure 1-1 show that the anti-C19-PP antibody (HL-PP-0-rFc) specifically binds to the C19 mispair PP, but its binding strength is weak and it cannot strongly recognize the C1 and C3 mispairs. The results in Figure 1-2 also show that the anti-C19-NN antibody (HL-NN-0-rFc) specifically binds to the C1, C3, and C19 mispair NN, but also shows some binding to the correct H(-)L(+) pair for C1 and C3.

[0146] Example 4: Optimization of HL-PP-0 and HL-NN-0 by Amino Acid Modifications. Amino acid modifications were introduced into the variable region sequences of HL-PP-0 to improve its affinity for mispairs and to confer cross-reactivity to C1 and C3 mispairs. HL-NN-0 was modified to suppress cross-reactivity to correct pairs and enhance its specificity for mispairs. Comprehensive single-residue substitutions were introduced into all CDR sequences and some FR sequences of each antibody, and the binding of each variant to various antigen-specific antibodies was confirmed. Effective modifications were combined over multiple rounds to confirm binding ability. Amino acid modifications to each antibody and antibody preparation were performed using methods known to those skilled in the art. The binding activity of the optimized antibodies was measured using a method similar to that described in Example 3.2. The names, sequence numbers, and information on the amino acid modifications introduced for the optimized antibodies are listed in Table 4-1. The amino acid sequences of the CDRs of the optimized antibodies are listed in Table 4-2. Furthermore, binding sensorgrams for each optimized antibody are shown in Figures 1-3 to 1-12 and Figures 2-1 to 2-7.

[0147] In addition, some of the antibodies in Table 4-1 have an AviTag tag attached to the C-terminus of the heavy chain for use in the analyses in Example 5 and later. TM A construct was created with a peptide sequence added. TM For antibodies conjugated with AviTag, biotin ligase (BirA) (SEQ ID NO: 50) was co-expressed at an antibody:BirA plasmid mass ratio of 9:1, and biotin was added to the medium at a final concentration of 100 μM. Antibodies with biotin conjugated to the AviTag are designated as Avi in ​​Table 4-1.

[0148]

[0149]

[0150] Example 5: Establishment of a Detection Method for Light Chain Exchangers Example 5-1: Preparation of Sample Antibodies Containing Light Chain Exchangers for Detection Tests To conduct a detection test for light chain exchangers, sample antibodies containing each mispair and correct pair were prepared. Human IgG1 kappa antibodies, Tocilizumab (MRA) and Trastuzumab (HER), were used as templates to prepare the antibodies for the detection test. The names and sequence numbers of each antibody are shown in Table 5. Antibodies consisting of a single heavy chain and a single light chain were prepared using methods known to those skilled in the art. To generate antibodies consisting of three or more heavy chains and three or more light chains, parent antibodies containing the desired Fab combination were first prepared by two-chain expression. Equal amounts of the two parent antibodies were then mixed and subjected to reduction treatment. The target sample antibody was prepared by reconstituting them and then removing the reducing agent by dialysis or other methods. The intended combination of the prepared sample antibodies was confirmed by ion exchange chromatography.

[0151]

[0152] Example 5-2: Establishment of a detection method for light chain exchangers. Light chain exchangers were detected using GyroLab® xP (Gyros Protein Technologies AB) and Gyrolab® xPand (Gyros Protein Technologies AB). PBS-T (Sigma, P-3563) was used as the mobile phase. Antibodies prepared in Example 4 (HL-NN-0-rFc for antibodies with C1 modification, HL-NN-1-rFc for antibodies with C3 modification, and HL-NN-0-rFc for antibodies with C19 modification) were biotinylated or biotinylated during antibody expression (C1: HL-NN-0-rFc-Avi) and diluted to 100 μg / mL in PBS-T. These were used as capture molecules for ligands with the corresponding amino acid modifications (C1, C3, and C19). The antibodies prepared in Example 4 (HL-PP-13-rFc for antibodies with C1 modification, HL-PP-1-rFc for antibodies with C3 modification, and HL-PP-1-rFc for antibodies with C19 modification) were labeled with AlexaFluor 647 and diluted to 2.5 μg / mL in REXXIP F buffer (Gyros Protein Technologies AB, P0004825) to be used as detection molecules for antibodies with the corresponding amino acid modifications (C1, C3, C19). The antigen-use antibodies with the corresponding amino acid modifications (C1, C3, C19) prepared in Example 5-1 were diluted with PBS-T and used as calibration curve samples or unknown samples. The concentrations of the calibration curve samples are shown in Table 6. Gyrolab Bioaffy TM 200 CD (Gyros Protein Technologies AB, P0004180) or Gyrolab Bioaffy TMLigand capture molecules were added to a 1000HC CD (Gyros Protein Technologies AB, P0020245), and the measurement sample was then captured. Detection molecules were then added to detect the sample captured on the CD. The values ​​detected for the calibration sample were processed with 4-PLA using Gyrolab evaluation software to create a calibration curve, which was then used to calculate the quantitative values ​​of light chain exchangers in unknown samples. The calibration curves are shown in Figures 3-1 to 3-7.

[0153]

[0154] Example 5-3: Quantification of the content of light chain-exchanged antibody in a mixed sample of MRA / HER antibody and light chain-exchanged antibody. For the corresponding amino acid modifications (C1, C3, C19), the antigen antibodies MRAH(N)MRAL(P) / HERH(P)HERL(N), MRAH(N)MRAL(P), and HERH(P)HERL(N) prepared in Example 5-1 were added with light chain-exchanged antibody (MRAH(N)HERL(N) / HERH(P)MRAL(P)) (NNPP in the table), and the concentration was adjusted with PBS-T. Analysis was performed using the method of Example 5-2. The recovery rate of the light chain-exchanged antibody was calculated from the quantitative values ​​of the light chain-exchanged antibody obtained. The results confirmed that the CV was 30% or less and the spike recovery was within 100 ± 30% under each condition. It is believed that light chain-exchanged antibody can also be similarly quantified using antibodies that specifically recognize mispairing between heavy and light chains, even with pairs other than the detection antibodies used in this study.

[0155]

[0156]

[0157]

[0158]

[0159] Example 5-4: Quantification of the content of light chain-exchanged antibodies in a mixed sample of bispecific antibodies other than MRA / HER and their light chain-exchanged antibodies. To confirm that the light chain-exchanged antibody quantification system can be used universally for antibodies with C1 and C3 modifications, 5% of the corresponding light chain-exchanged antibody (Lch-exchanged antibody in Table 11) was added to the tested sample listed in Table 11 and analyzed by the method of Example 5-2 (the corresponding sequence numbers are listed in Table 5, and the calibration curves are shown in Figures 3-5, 3-6, and 3-7). The recovery rate of the light chain-exchanged antibody was calculated from the quantitative values ​​obtained. The results confirmed that the CV was 30% or less and the spike recovery was within 100 ± 30% under each condition. These results demonstrate that the light chain-exchanged antibody quantification method described in this invention can be used universally, regardless of the antibody's Fab sequence.

[0160]

[0161] The method of the present disclosure, which uses an antibody that recognizes mispaired variants of a bispecific antibody, makes it possible to accurately quantify light chain exchange products contained as impurities during the production of a bispecific antibody.

Claims

1. A method for quantifying or detecting mispaired variants of a multispecific antigen-binding molecule, wherein the mispaired variants comprise at least two different types of F(ab) that are not present in the multispecific antigen-binding molecule, the method comprising: (a) contacting a first detection antibody with a sample containing the mispaired variant; (b) contacting a second detection antibody with the mispaired variant bound to the first detection antibody; and (c) quantifying or detecting the second detection antibody bound to the mispaired variant, wherein one of the first or second detection antibodies binds to one of the F(ab), and the other binds to the other F(ab).

2. The method of claim 1, wherein the mispaired variant comprises a first and a second F(ab), wherein the first F(ab) has at least a first pair of amino acid residues forming a VH-VL interface or a CH1-CL interface, and both amino acid residues forming the first pair of amino acid residues are positively charged, and the second F(ab) has at least a second pair of amino acid residues forming a VH-VL interface or a CH1-CL interface, and both amino acid residues forming the second pair of amino acid residues are negatively charged, and one of the first or second detection antibodies binds to one of the first or second F(ab), and the other binds to the other of the first or second F(ab).

3. The method of claim 1, wherein the mispair variants include: (1) a first and a second F(ab) in which the amino acids at positions 147 and 175 in CH1 and the amino acids at positions 131 and 160 in CL are charge-controlled to repel each other; (2) a first and a second F(ab) in which the amino acids at positions 147 and 175 in CH1 and the amino acids at positions 131 and 180 in CL are charge-controlled to repel each other; or (3) a first and a second F(ab) in which the amino acids at positions 147 and 175 in CH1 and the amino acids at positions 131, 160, and 180 in CL are charge-controlled to repel each other; wherein the position numbers of CH1 are according to EU numbering and the position numbers of CL are according to Kabat numbering.

4. The method of claim 1, wherein the mispaired variant F(ab) comprises the following amino acid residues: (1) K at positions 147 and 175 in CH1; and K at positions 131 and 160 in CL; (2) K at positions 147 and 175 in CH1; and K at positions 131 and 180 in CL; (3) K at positions 147 and 175 in CH1; and K at positions 160 and 180 in CL; (4) K at positions 147 and 213 in CH1; and K at positions 123 and 131 in CL; (5) K at positions 147 and 213 in CH1; and K at positions 123 and 160 in CL; (6) K at positions 147 and 213 in CH1; and K at positions 123 and 180 in CL; (7) K at positions 175 and 213 in CH1; and K at positions 123 and 131 in CL; (8) K at positions 175 and 213 in CH1; and K at positions 123 and 160 in CL; (9) K at positions 175 and 213 in CH1; and K at positions 123 and 180 in CL; (10) K at positions 147 and 175 in CH1; and K at positions 131, 160, and 180 in CL; (11) K at positions 147, 175, and 213 in CH1; and K at positions 123, 131, 160, and 180 in CL; where the position numbers in CH1 follow EU numbering, and the position numbers in CL follow Kabat numbering.

5. The method of claim 1, wherein the mispaired variant F(ab) comprises the following amino acid residues: (1) E at positions 147 and 175 in CH1; and E at positions 131 and 160 in CL; (2) E at positions 147 and 175 in CH1; and E at positions 131 and 180 in CL; (3) E at positions 147 and 175 in CH1; and E at positions 160 and 180 in CL; (4) E at positions 147 and 213 in CH1; and E at positions 123 and 131 in CL; (5) E at positions 147 and 213 in CH1; and E at positions 123 and 160 in CL; (6) E at positions 147 and 213 in CH1; and E at positions 123 and 180 in CL; (7) E at positions 175 and 213 in CH1; and E at positions 123 and 131 in CL; (8) E at positions 175 and 213 in CH1; and E at positions 123 and 160 in CL; (9) E at positions 175 and 213 in CH1; and E at positions 123 and 180 in CL; (10) E at positions 147 and 175 in CH1; and E at positions 131, 160, and 180 in CL; (11) E at positions 147, 175, and 213 in CH1; and E at positions 123, 131, 160, and 180 in CL; where the position numbers in CH1 follow EU numbering and the position numbers in CL follow Kabat numbering.

6. The method of claim 1, wherein the first detection antibody is biotinylated.

7. The method of claim 1, wherein the second detection antibody is fluorescently labeled.

8. An antibody that binds to an F(ab) present in a mispaired variant of a multispecific antigen-binding molecule, but does not bind to an F(ab) present in the multispecific antigen-binding molecule.

9. The antibody described in claim 8, wherein the mispaired variant F(ab) has at least one pair of amino acid residues forming the VH-VL interface or the CH1-CL interface, and both amino acid residues forming the amino acid residue pair have the same charge.

10. The antibody of claim 8, which binds to the following F(ab): (1) an F(ab) in which the charge is controlled so that the amino acids at positions 147 and 175 in CH1 and the amino acids at positions 131 and 160 in CL repel each other; (2) an F(ab) in which the charge is controlled so that the amino acids at positions 147 and 175 in CH1 and the amino acids at positions 131 and 180 in CL repel each other; or (3) an F(ab) in which the charge is controlled so that the amino acids at positions 147 and 175 in CH1 and the amino acids at positions 131, 160, and 180 in CL repel each other; wherein the position numbers of CH1 are according to EU numbering and the position numbers of CL are according to Kabat numbering.

11. The antibody of claim 8, which binds to an F(ab) containing the following amino acid residues present in a mispair variant: (1) K at positions 147 and 175 in CH1; and K at positions 131 and 160 in CL; (2) K at positions 147 and 175 in CH1; and K at positions 131 and 180 in CL; (3) K at positions 147 and 175 in CH1; and K at positions 160 and 180 in CL; (4) K at positions 147 and 213 in CH1; and K at positions 123 and 131 in CL; (5) K at positions 147 and 213 in CH1; and K at positions 123 and 160 in CL; (6) K at positions 147 and 213 in CH1; (7) K at positions 175 and 213 in CH1; and K at positions 123 and 131 in CL; (8) K at positions 175 and 213 in CH1; and K at positions 123 and 160 in CL; (9) K at positions 175 and 213 in CH1; and K at positions 123 and 180 in CL; (10) K at positions 147 and 175 in CH1; and K at positions 131, 160, and 180 in CL; (11) K at positions 147, 175, and 213 in CH1; and K at positions 123, 131, 160, and 180 in CL; Here, the position numbers of CH1 follow EU numbering, and the position numbers of CL follow Kabat numbering.

12. The antibody of claim 8, selected from the following: (1) an antibody capable of binding to an F(ab) in which positions 147 and 175 in CH1 are K and positions 131 and 160 in CL are K, but an F(ab) in which positions 147 and 175 in CH1 are K and positions 131 and 160 in CL are E; or an antibody incapable of binding to an F(ab) in which positions 147 and 174 in CH1 are E and positions 131 and 160 in CL are K; (2) an antibody capable of binding to an F(ab) in which positions 147 and 175 in CH1 are K and positions 131 and 180 in CL, but an F(ab) in which positions 147 and 175 in CH1 are K and positions 131 and 180 in CL are E; or an antibody that cannot bind to an F(ab) in which positions 147 and 175 in CH1 are E and positions 131 and 180 in CL are K; (3) an antibody that can bind to an F(ab) in which positions 147 and 175 in CH1 are K and positions 160 and 180 in CL are K, but an F(ab) in which positions 147 and 175 in CH1 are K and positions 160 and 180 in CL are E; or an antibody that cannot bind to an F(ab) in which positions 147 and 175 in CH1 are E and positions 160 and 180 in CL; (4) an antibody that can bind to an F(ab) in which positions 147 and 213 in CH1 are K and positions 123 and 131 in CL, but (5) An antibody that can bind to an F(ab) in which positions 147 and 213 in CH1 are K and positions 123 and 131 in CL are K; or an antibody that cannot bind to an F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 131 in CL are K; (6) An antibody that can bind to an F(ab) in which positions 147 and 213 in CH1 are K and positions 123 and 160 in CL are K, but cannot bind to an F(ab) in which positions 147 and 213 in CH1 are K and positions 123 and 160 in CL are K; or an antibody that cannot bind to an F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 160 in CL;(6) An antibody capable of binding to F(ab) in which positions 147 and 213 in CH1 are K and positions 123 and 180 in CL are K, but in which positions 147 and 213 in CH1 are K and positions 123 and 180 in CL are E; or in which positions 147 and 213 in CH1 are E and positions 123 and 180 in CL are K; (7) An antibody capable of binding to F(ab) in which positions 175 and 213 in CH1 are K and positions 123 and 131 in CL are K, but in which positions 175 and 213 in CH1 are K and positions 123 and 131 in CL are E; or an antibody that cannot bind to an F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 131 in CL are K; (8) an F(ab) in which positions 175 and 213 in CH1 are K and positions 123 and 160 in CL are K, but an F(ab) in which positions 175 and 213 in CH1 are K and positions 123 and 131 in CL are E; or an antibody that cannot bind to an F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 131 in CL are K;(9) An antibody capable of binding to F(ab) in which positions 175 and 213 in CH1 are K and positions 123 and 180 in CL are K, but in which positions 175 and 213 in CH1 are K and positions 123 and 180 in CL are E; or an antibody incapable of binding to F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 180 in CL are K; (10) An antibody capable of binding to F(ab) in which positions 147 and 175 in CH1 are K and positions 131, 160, and 180 in CL are K, but in which positions 147 and 175 in CH1 are K and positions 131, 160, and 180 in CL are E; or (11) an antibody that can bind to an F(ab) in which positions 147, 175, and 213 in CH1 are K and positions 123, 131, 160, and 180 in CL are K, but an F(ab) in which positions 147, 175, and 213 in CH1 are K and positions 123, 131, 160, and 180 in CL are E; or an antibody that cannot bind to an F(ab) in which positions 147, 175, and 213 in CH1 are E and positions 123, 131, 160, and 180 in CL are K; Here, the position numbers of CH1 follow EU numbering, and the position numbers of CL follow Kabat numbering.

13. The antibody of claim 8, which binds to an F(ab) containing the following amino acid residues present in a mispair variant: (1) E at positions 147 and 175 in CH1; and E at positions 131 and 160 in CL; (2) E at positions 147 and 175 in CH1; and E at positions 131 and 180 in CL; (3) E at positions 147 and 175 in CH1; and E at positions 160 and 180 in CL; (4) E at positions 147 and 213 in CH1; and E at positions 123 and 131 in CL; (5) E at positions 147 and 213 in CH1; and E at positions 123 and 160 in CL; (6) E at positions 147 and 213 in CH1; and E at positions 123 and 180 in CL; (7) E at positions 175 and 213 in CH1; and E at positions 123 and 131 in CL; (8) E at positions 175 and 213 in CH1; and E at positions 123 and 160 in CL; (9) E at positions 175 and 213 in CH1; and E at positions 123 and 180 in CL; (10) E at positions 147 and 175 in CH1; and E at positions 131, 160, and 180 in CL; (11) E at positions 147, 175, and 213 in CH1; and E at positions 123, 131, 160, and 180 in CL; Here, the position numbers of CH1 follow EU numbering, and the position numbers of CL follow Kabat numbering.

14. The antibody of claim 8, selected from the following: (1) an antibody capable of binding to an F(ab) having E at positions 147 and 175 in CH1 and E at positions 131 and 160 in CL, but not to an F(ab) having E at positions 147 and 175 in CH1 and E at positions 131 and 160 in CL; or an antibody that does not bind to an F(ab) having E at positions 147 and 174 in CH1 and K at positions 131 and 160 in CL; (2) an antibody capable of binding to an F(ab) having E at positions 147 and 175 in CH1 and E at positions 131 and 180 in CL, but not to an F(ab) having K at positions 147 and 175 in CH1 and E at positions 131 and 180 in CL; or an antibody that cannot bind to F(ab) in which positions 147 and 175 in CH1 are E and positions 131 and 180 in CL are K; (3) an antibody that can bind to F(ab) in which positions 147 and 175 in CH1 are E and positions 160 and 180 in CL are E, but an F(ab) in which positions 147 and 175 in CH1 are K and positions 160 and 180 in CL are E; or an antibody that cannot bind to F(ab) in which positions 147 and 175 in CH1 are E and positions 160 and 180 in CL; (4) an antibody that can bind to F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 131 in CL are E, but (5) An antibody capable of binding to an F(ab) in which positions 147 and 213 in CH1 are K and positions 123 and 131 in CL are K; (6) An antibody capable of binding to an F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 131 in CL are K; (7) An antibody capable of binding to an F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 160 in CL are E; or (8) An antibody capable of binding to an F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 160 in CL are K; or (9) An antibody capable of binding to an F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 160 in CL are K;(6) An antibody capable of binding to F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 180 in CL are E, but an F(ab) in which positions 147 and 213 in CH1 are K and positions 123 and 180 in CL are E; or an antibody incapable of binding to F(ab) in which positions 147 and 213 in CH1 are E and positions 123 and 180 in CL are K; (7) An antibody capable of binding to F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 131 in CL are E, but an F(ab) in which positions 175 and 213 in CH1 are K and positions 123 and 131 in CL are E; or an antibody that cannot bind to an F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 131 in CL are K; (8) an F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 160 in CL are E, but an F(ab) in which positions 175 and 213 in CH1 are K and positions 123 and 131 in CL; or an antibody that cannot bind to an F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 131 in CL are K;(9) An antibody capable of binding to F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 180 in CL are E, but in which positions 175 and 213 in CH1 are K and positions 123 and 180 in CL are E; or an antibody incapable of binding to F(ab) in which positions 175 and 213 in CH1 are E and positions 123 and 180 in CL are K; (10) An antibody capable of binding to F(ab) in which positions 147 and 175 in CH1 are E and positions 131, 160, and 180 in CL are E, but in which positions 147 and 175 in CH1 are K and positions 131, 160, and 180 in CL are E; or (11) an antibody capable of binding to an F(ab) in which positions 147, 175, and 213 in CH1 are E and positions 123, 131, 160, and 180 in CL are E, but an F(ab) in which positions 147, 175, and 213 in CH1 are E and positions 123, 131, 160, and 180 in CL are E; or an antibody incapable of binding to an F(ab) in which positions 147, 175, and 213 in CH1 are E and positions 123, 131, 160, and 180 in CL are K; wherein the CH1 position numbers are according to EU numbering and the CL position numbers are according to Kabat numbering. ; 15. The antibody of claim 8, comprising the following VH and VL: (1) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO:64, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO:69, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO:75; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO:80, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO:85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO:90; (2) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO:65, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO:70, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO:76; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO:81, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO:85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO:91; (3) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO:65, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO:70, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO:76; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 86, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 91; (4) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 70, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 76; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 92; (5) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 70, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 77; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 92;(6) VH comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 70, and HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 77; and VL comprising LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 86, and LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 92; (7) VH comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 70, and HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 77; and VL comprising LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 93; (8) VH comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 70, and HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 77; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 81, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 92; (9) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 66, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 71, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 77; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 87, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 94; (10) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 66, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 71, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 77; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 93;(11) VH comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 72, and HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 78; and VL comprising LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 93; (12) VH comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 71, and HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 78; and VL comprising LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 93; (13) VH comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 66, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 71, and HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 78; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 93; (14) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 66, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 73, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 77; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 82, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 93; (15) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 67, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 74, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 79; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 83, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 88, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 95;(16) VH comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 67, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 74, and HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 79; and VL comprising LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 84, LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 88, and LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 95; (17) VH comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 67, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 74, and HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 79; and VL comprising LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 83, LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 89, and LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 95; (18) VH comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 68, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 74, and HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 79; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 84, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 88, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 95; or (19) a VH comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 68, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 74, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 79; and a VL comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 83, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 89, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO:

96.

16. The antibody of claim 8, comprising the following VH and VL: (1) a VH comprising the amino acid sequence set forth in SEQ ID NO: 8 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 31; (2) a VH comprising the amino acid sequence set forth in SEQ ID NO: 8 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 32; (3) a VH comprising the amino acid sequence set forth in SEQ ID NO: 8 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 33; (4) a VH comprising the amino acid sequence set forth in SEQ ID NO: 9 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 33; (5) a VH comprising the amino acid sequence set forth in SEQ ID NO: 9 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 34; (6) a VH comprising the amino acid sequence set forth in SEQ ID NO: 9 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (7) a VH comprising the amino acid sequence set forth in SEQ ID NO: 9 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 36; (8) a VH comprising the amino acid sequence set forth in SEQ ID NO: 10 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 37; (9) VH comprising the amino acid sequence set forth in SEQ ID NO: 10 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (10) VH comprising the amino acid sequence set forth in SEQ ID NO: 11 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (11) VH comprising the amino acid sequence set forth in SEQ ID NO: 12 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (12) VH comprising the amino acid sequence set forth in SEQ ID NO: 13 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (13) VH comprising the amino acid sequence set forth in SEQ ID NO: 14 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (14) VH comprising the amino acid sequence set forth in SEQ ID NO: 9 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (15) VH comprising the amino acid sequence set forth in SEQ ID NO: 10 and VL comprising the amino acid sequence set forth in SEQ ID NO: 37; (16) VH comprising the amino acid sequence set forth in SEQ ID NO: 10 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (17) VH comprising the amino acid sequence set forth in SEQ ID NO: 11 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35;(18) VH comprising the amino acid sequence set forth in SEQ ID NO: 12 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (19) VH comprising the amino acid sequence set forth in SEQ ID NO: 13 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (20) VH comprising the amino acid sequence set forth in SEQ ID NO: 14 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (21) VH comprising the amino acid sequence set forth in SEQ ID NO: 9 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (22) VH comprising the amino acid sequence set forth in SEQ ID NO: 10 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (23) VH comprising the amino acid sequence set forth in SEQ ID NO: 11 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (24) VH comprising the amino acid sequence set forth in SEQ ID NO: 12 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (25) VH comprising the amino acid sequence set forth in SEQ ID NO: 13 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (26) VH comprising the amino acid sequence set forth in SEQ ID NO: 14 and VL comprising the amino acid sequence set forth in SEQ ID NO: 35; (27) VH comprising the amino acid sequence set forth in SEQ ID NO: 7 and VL comprising the amino acid sequence set forth in SEQ ID NO: 38; (28) VH comprising the amino acid sequence set forth in SEQ ID NO: 7 and VL comprising the amino acid sequence set forth in SEQ ID NO: 39; (29) VH comprising the amino acid sequence set forth in SEQ ID NO: 15 and VL comprising the amino acid sequence set forth in SEQ ID NO: 38; (30) VH comprising the amino acid sequence set forth in SEQ ID NO: 15 and VL comprising the amino acid sequence set forth in SEQ ID NO: 40; (31) VH comprising the amino acid sequence set forth in SEQ ID NO: 7 and VL comprising the amino acid sequence set forth in SEQ ID NO: 30; (32) VH comprising the amino acid sequence set forth in SEQ ID NO: 6 and VL comprising the amino acid sequence set forth in SEQ ID NO: 29; or (33) VH comprising the amino acid sequence set forth in SEQ ID NO: 7 and VL comprising the amino acid sequence set forth in SEQ ID NO:

30.

17. A nucleic acid encoding an antibody, or antigen-binding fragment thereof, according to any one of claims 8 to 16.

18. A host cell comprising the nucleic acid of claim 17.

19. A method for producing an antibody for detecting mispaired variants, comprising a step of immunizing with an F(ab) present in a mispaired variant of a multispecific antigen-binding molecule, wherein the F(ab) has at least one pair of amino acid residues forming a VH-VL interface or a CH1-CL interface, and both amino acid residues forming the amino acid residue pair have the same charge.

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