Library of antigen-binding molecules comprising a modified antibody variable region

Abstract

To provide a library that consists of a plurality of antigen-binding molecules having different sequences from each other, and includes a variable region of an antibody, in which the variable region has binding activity to a first antigen and a second antigen different from the first antigen, but does not simultaneously bind to the first antigen and the second antigen.SOLUTION: A library that mainly consists of a plurality of antigen-binding molecules having different sequences from each other, in which an antigen-binding region in the antigen-binding molecule is a variable region of an antibody which can bind to the first antigen and a second antigen different from the first antigen, but does not simultaneously bind to the first antigen and the second antigen, either one of the first antigen and the second antigen is CD3, and the other antigen is a molecule expressed on the surface of a T-cell or other immunocyte.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention provides a population (library) of antigen-binding molecules comprising variable regions of antibodies that can bind to two different antigens (a first antigen and a second antigen) but do not bind to both antigens simultaneously, a method for producing the library, a method for producing a bispecific antibody comprising a common light chain variable region using the library, and a method for selecting an antigen-binding molecule comprising a variable region with enhanced binding to a first antigen. The present invention also relates to an antigen-binding molecule comprising variable regions of antibodies that can bind to three different antigens (a first antigen, a second antigen, and a fourth antigen) but do not bind to the three antigens simultaneously, a pharmaceutical composition comprising the antigen-binding molecule, and methods for producing them.

Background Art

[0002] Antibodies have attracted attention as pharmaceuticals because of their high stability in plasma and few side effects (Nat. Biotechnol. (2005) 23, 1073-1078 (Non-Patent Document 1) and Eur J Pharm Biopharm. (2005) 59 (3), 389-396 (Non-Patent Document 2)). Antibodies not only act to bind to antigens, agonistic or antagonistic actions, but also induce cytotoxic activities (also referred to as effector functions) by effector cells such as ADCC (Antibody Dependent Cytotoxicity), ADCP (Antibody Dependent Cell phagocytosis), and CDC (complement-dependent cytotoxic activity). In particular, antibodies of the IgG1 subclass exhibit effector functions against cancer cells, and thus many antibody pharmaceuticals have been developed in the cancer field.

[0003] For an antibody to exhibit ADCC, ADCP, or CDC, it is essential for the Fc region of the antibody to bind to antibody receptors (FcγRs) present on effector cells such as NK cells and macrophages, as well as various complement components. In humans, isoforms of FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb have been reported as the protein family of FcγRs, and their respective allotypes have also been reported (Immunol. Lett. (2002) 82, 57-65 (Non-Patent Document 3)). Among these isoforms, FcγRIa, FcγRIIa, and FcγRIIIa have a domain called ITAM (Immunoreceptor Tyrosine-based Activation Motif) in their intracellular domain and transmit activation signals. On the other hand, only FcγRIIb has a domain called ITIM (Immunoreceptor Tyrosine-based Inhibitory Motif) in its intracellular domain and transmits inhibitory signals. It is known that any FcγR transmits signals when cross-linked by an immune complex or the like (Nat. Rev. Immunol. (2008) 8, 34-47 (Non-Patent Document 4)). In fact, when an antibody exerts an effector function on cancer cells, the FcγRs on the effector cell membrane cluster at the Fc regions of multiple antibodies bound on the cancer cell membrane, and activation signals are transmitted in the effector cells. As a result, a cytotoxic effect is exerted. At this time, since the cross-linking of FcγR is limited to effector cells present in the vicinity of cancer cells, it indicates that immune activation occurs only locally at the cancer cells. (Ann. Rev. Immunol. (1988). 6. 251-81 (Non-Patent Document 5))

[0004] Natural immunoglobulins bind to antigens in the variable region and to receptors such as FcγR, FcRn, FcαR, and FcεR and complement in the constant region. FcRn, which is one of the binding molecules that interact in the Fc region of IgG, binds one molecule each to the heavy chains of the antibody, and it has been reported that two molecules of FcRn bind to one molecule of IgG-type antibody. However, unlike FcRn and others, FcγR interacts with the hinge region and CH2 domain of the antibody and binds only one molecule to one molecule of IgG-type antibody (J. Bio. Chem., (20001) 276, 16469-16477). Also, for the binding of FcγR to the Fc region of the antibody, it has been shown that several amino acid residues in the hinge region and CH2 domain of the antibody and the sugar chain added to Asn at position 297 of the EU numbering that binds to the CH2 domain are important (Chem. Immunol. (1997), 65, 88-110 (Non-Patent Document 6), Eur. J. Immunol. (1993) 23, 1098-1104 (Non-Patent Document 7), Immunol. (1995) 86, 319-324 (Non-Patent Document 8)). Centering around this binding site, various mutants of the Fc region with different FcγR binding properties have been studied so far, and Fc region mutants with higher binding activity to activating FcγR have been obtained (WO2000 / 042072 (Patent Document 1), WO2006 / 019447 (Patent Document 2)). For example, Lazar et al. succeeded in increasing the binding activity of human IgG1 to human FcγRIIIa (V158) by about 370-fold by substituting Ser at position 239, Ala at position 330, and Ile at position 332 of human IgG1 with Asn, Leu, and Glu, respectively (Proc. Natl. Acad. Sci. U. S. A. (2006) 103, 4005-4010 (Non-Patent Document 9), WO2006 / 019447 (Patent Document 2)). This variant has an approximately 9-fold ratio of binding activity to FcγRIIIa to FcγIIb (A / I ratio) compared to the wild type.Moreover, Shinkawa et al. have succeeded in increasing the binding activity to FcγRIIIa by about 100-fold by deleting the fucose of the sugar chain added to the 297th Asn of the EU numbering (J. Biol. Chem. (2003) 278, 3466-3473 (Non-Patent Document 10)). By these methods, it is possible to significantly improve the ADCC activity of human IgG1 as compared with natural human IgG1.

[0005] Normal natural IgG-type antibodies recognize and bind only one epitope by their variable regions (Fab), and thus can bind to only one antigen. On the other hand, in cancer and inflammation, it is known that multiple types of proteins are involved, and there are cases where proteins interact with each other. For example, in immune diseases, it is known that several inflammatory cytokines (TNF, IL1, and IL6) are involved (Nat. Biotech., (2011) 28, 502-10 (Non-Patent Document 11)). Also, as one of the mechanisms for acquiring drug resistance in cancer, it is known that other receptors are activated (Endocr Relat Cancer (2006) 13, 45-51 (Non-Patent Document 12)). In such cases, normal antibodies that recognize one epitope cannot inhibit multiple proteins.

[0006] As a molecule that inhibits multiple targets, antibodies that bind to two or more types of antigens with a single molecule (referred to as bispecific antibodies) have been studied. By modifying natural IgG-type antibodies, it is possible to confer binding activity to two different antigens (the first antigen and the second antigen) (MAbs. (2012) Mar 1, 4(2)). Therefore, in addition to the action of neutralizing two or more types of antigens with a single molecule, there is an action of enhancing antitumor activity by cross-linking cells with cytotoxic activity and cancer cells. So far, as molecular forms of bispecific antibodies, molecules with antigen-binding sites added to the N-terminus or C-terminus of the antibody (DVD-Ig and scFv-IgG), molecules in which the two Fab regions of the antibody have different sequences (common light chain bispecific antibodies and hybrid hybridomas), molecules in which one Fab region recognizes two antigens (Two-in-one IgG), and molecules with a loop site in the CH3 region as a new antigen-binding site (Fcab) have been reported (Nat. Rev. (2010), 10, 301-316 (Non-Patent Document 13), Peds(2010), 23(4), 289-297 (Non-Patent Document 14)). Since all bispecific antibodies interact with FcγR in the Fc region, the effector function of the antibody is preserved. Therefore, for any antigen recognized by the bispecific antibody, it binds to FcγR at the same time and exhibits ADCC activity against cells expressing the antigen.

[0007] If all the antigens recognized by the bispecific antibody are antigens specifically expressed in cancer, binding to any of the antigens will show cytotoxic activity against cancer cells, so a more efficient anti-cancer effect can be expected than that of a normal antibody drug that recognizes a single antigen. However, if any one of the antigens recognized by the bispecific antibody is expressed in normal tissue or is a cell expressed in immune cells, cross-linking with FcγR will cause damage to normal tissue and release of cytokines (J. Immunol. (1999) Aug 1, 163(3), 1246-52 (Non-Patent Document 15)). As a result, strong side effects will be induced.

[0008] For example, Catumaxomab is known as a bispecific antibody that recognizes a protein expressed on T cells and a protein expressed on cancer cells (cancer antigen). Catumaxomab binds to the cancer antigen (EpCAM) and the CD3ε chain expressed on T cells with two Fabs, respectively. By simultaneously binding the cancer antigen and CD3ε, Catumaxomab induces cytotoxic activity by T cells, and by simultaneously binding the cancer antigen and FcγR, it induces cytotoxic activity by antigen-presenting cells such as NK cells and macrophages. By utilizing these two cytotoxic activities, Catumaxomab has shown a high therapeutic effect in malignant ascites by intraperitoneal administration and has been approved in Europe. (Cancer Treat Rev. (2010) Oct 36(6), 458-67 (Non-Patent Document 16)) Furthermore, an example has been reported in which an antibody reactive to cancer cells appears after administration of Catumaxomab, and it has been clarified that acquired immunity is induced. (Future Oncol. (2012) Jan 8(1), 73-85 (Non-Patent Document 17)). From these results, antibodies having both the cytotoxic activity by T cells and the action by cells such as NK cells and macrophages via FcγR (especially called trifunctional antibodies) are attracting attention because strong antitumor effects and induction of acquired immunity can be expected.

[0009] However, since trifunctional antibodies simultaneously bind CD3ε and FcγR even in the absence of a cancer antigen, T cells expressing CD3ε and cells expressing FcγR are cross-linked even in an environment where cancer cells are absent, and various cytokines are produced in large amounts. Due to the induction of production of various cytokines independent of the cancer antigen, administration of trifunctional antibodies is currently limited to intraperitoneal use (Cancer Treat Rev. 2010 Oct 36(6), 458-67 (Non-Patent Document 16)), and systemic administration is extremely difficult due to severe cytokine storm-like side effects (Cancer Immunol Immunother. 2007 Sep;56(9):1397-406 (Non-Patent Document 18)). In addition, in the bispecific antibodies of the prior art, since both antigens, the cancer antigen (EpCAM) as the first antigen and CD3ε as the second antigen, can bind to FcγR simultaneously, it is impossible structurally to avoid such side effects caused by the simultaneous binding of FcγR and the second antigen CD3ε.

[0010] In recent years, by using an Fc region with reduced binding activity to FcγR, improved antibodies that can cause cytotoxic activity by T cells while avoiding side effects have been provided (WO2012 / 073985). However, even with such antibodies, structurally, it is impossible to act on two immune receptors, CD3ε and FcγR, while binding to the cancer antigen. So far, no antibody has been known that can act on both the cytotoxic activity by T cells and the cytotoxic activity by cells other than T cells specifically for cancer antigens while avoiding side effects.

Prior Art Documents

Patent Documents

[0011]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0012]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 18

Summary of the Invention

Problems to be Solved by the Invention

[0013] The present invention has been made in view of such circumstances, and the problems thereof are a population (library) of antigen-binding molecules containing variable regions of antibodies that have binding activity against two different antigens (a first antigen and a second antigen) but do not bind to these antigens simultaneously, a method for producing the library, a method for selecting or producing a desired antigen-binding molecule using the library, and a method for selecting an antigen-binding molecule containing a variable region with enhanced binding to a first antigen. Further, the problems of the present invention are to provide an antigen-binding molecule containing a variable region of an antibody that can bind to three different antigens (a first antigen, a second antigen, and a fourth antigen) but does not bind to these three antigens simultaneously, a pharmaceutical composition containing the antigen-binding molecule, and methods for producing them.

Means for Solving the Problems

[0014] The inventors of the present invention have conducted intensive research to solve the above problems. As a result, the inventors have prepared an antigen-binding molecule comprising a variable region of an antibody that has binding activity to two different antigens (a first antigen and a second antigen) but does not bind to these antigens simultaneously, and a variable region that binds to an antigen (a third antigen) different from these antigens, and succeeded in enhancing the activity generated by the antigen-binding molecule by utilizing the binding activity of the antigen-binding molecule to three different antigens. Furthermore, when using a conventional multispecific antigen-binding molecule as a pharmaceutical, it has been successful in preparing an antigen-binding molecule capable of avoiding cross-linking between different cells caused by binding to antigens expressed on different cells, which is considered to be a cause of side effects. Furthermore, it has been successful in preparing a library of antigen-binding molecules containing a variable region of an antibody that can bind to two different antigens (a first antigen and a second antigen) but does not bind to both antigens simultaneously. In addition, using the library, it has been successful in obtaining (selecting) an antigen-binding molecule having binding activity to two desired antigens, and in obtaining (selecting) a variable region with enhanced binding to a desired antigen. Furthermore, the inventors have succeeded in preparing an antigen-binding molecule containing a variable region of an antibody that can bind to three different antigens (a first antigen, a second antigen, and a fourth antigen) but does not bind to these three antigens simultaneously.

[0015] More specifically, the present invention relates to the following. 〔1〕A library mainly composed of a plurality of antigen-binding molecules having different sequences, wherein the antigen-binding region in the antigen-binding molecule is a variable region of an antibody that can bind to a first antigen and a second antigen different from the first antigen, but does not bind to the first antigen and the second antigen simultaneously, and either one of the first antigen and the second antigen is CD3, and the other antigen is a molecule expressed on the surface of T cells or other immune cells. 〔2〕The library according to 〔1〕, wherein the variable region that does not bind to the first antigen and the second antigen simultaneously is a variable region that does not bind to the first antigen and the second antigen expressed on different cells simultaneously. 〔3〕The variable region is a variable region into which at least one amino acid modification has been introduced, and the modified amino acid is an amino acid in the CDR1, CDR2, CDR3, or FR3 region of the variable region of the antibody. The library according to 〔1〕 or 〔2〕. 〔4〕The modified amino acid is an amino acid at at least one position selected from Kabat numbering 31 - 35, 50 - 65, 71 - 74, and 95 - 102 of the heavy chain variable region of the antibody, and Kabat numbering 24 - 34, 50 - 56, and 89 - 97 of the light chain variable region. The library according to 〔3〕. 〔5〕The other antigen is an FcγR, TLR, lectin, IgA, immune checkpoint molecule, TNF superfamily molecule, TNFR superfamily molecule, or NK receptor molecule. The library according to any one of 〔1〕 to 〔4〕. 〔6〕The antigen - binding molecule is a fusion polypeptide of the variable region of an antibody and at least a part of a viral coat protein. The library according to any one of 〔1〕 to 〔5〕. 〔7〕The at least one amino acid modification has been introduced with respect to a template sequence consisting of the heavy chain variable region sequence described in SEQ ID NO: 96 and / or the light chain variable region sequence described in SEQ ID NO: 53, and the modified amino acid is an amino acid at any one or more positions selected from the following: The library according to any one of 〔3〕 to 〔6〕: Heavy chain: 31, 52b, 52c, 53, 54, 56, 57, 61, 98, 99, 100, 100a, 100b, 100c, 100d, 100e, 100f, 100g (Kabat numbering); Light chain: 24, 25, 26, 27, 27a, 27b, 27c, 27e, 30, 31, 33, 34, 51, 52, 53, 54, 55, 56, 74, 77, 89, 90, 92, 93, 94, 96 (Kabat numbering). 〔8〕The following steps (a) and (b): (a) Using the variable region sequence of an antibody that binds to a first antigen as the template sequence of the library, a step of identifying amino acid modifications that satisfy any one or more of the following (i) to (iii): (i) Modifications that do not substantially change the binding ability to the first antigen; (ii) Modifications that do not substantially change the ECM binding ability; and (iii) Insertion of a peptide consisting of 1 to 25 amino acids into the CDR1, CDR2, CDR3, or FR3 region of the heavy chain variable region; and (b) A step of designing a library comprising a nucleic acid encoding the template sequence and nucleic acids encoding variable regions having different sequences from each other and having one or more amino acid modifications identified in step (a) in the template sequence The method for producing a library according to any one of [1] to [7], comprising: [9] The method for producing a library according to [8], wherein the library is produced using the heavy chain variable region sequence described in SEQ ID NO: 96 and / or the light chain variable region sequence described in SEQ ID NO: 53 as the template sequence of the library.

[10] A method for producing an antigen-binding molecule comprising a variable region that can bind to a first antigen which is CD3 and a second antigen different from the first antigen but does not bind to the first antigen and the second antigen simultaneously: (a) A step of contacting the library according to any one of [1] to [7] with a second antigen; (b) A step of recovering the antigen-binding molecule bound to the second antigen in step (a); and (c) A step of selecting an antigen-binding molecule comprising a variable region that does not bind to the first antigen and the second antigen simultaneously from the population of antigen-binding molecules recovered in step (b).

[11] The method for producing an antigen-binding molecule according to

[10] , wherein the second antigen is an FcγR, TLR, lectin, IgA, immune checkpoint molecule, TNF superfamily molecule, TNFR superfamily molecule, or NK receptor molecule. A library mainly composed of a plurality of antigen-binding molecules with different arrangements, wherein the antigen-binding region in the antigen-binding molecule is a variable region of an antibody consisting of a template sequence or a variable region of an antibody having at least one amino acid modification in the template sequence, and can bind to a first antigen and a second antigen different from the first antigen, but is a variable region of an antibody that does not bind to the first antigen and the second antigen simultaneously. A method for producing a bispecific antibody, comprising the following steps (a) to (c): (a) A step of selecting the template sequence of the library as a variable region that binds to a first antigen; (b) A step of selecting a variable region that binds to a second antigen but does not bind to the first antigen as a variable region that binds to the second antigen, comprising the following steps (i) to (iv): (i) A step of contacting the library with a second antigen; (ii) A step of recovering the antigen-binding molecules bound to the second antigen in the step (i); (iii) A step of contacting the population of the antigen-binding molecules recovered in the step (ii) with the first antigen; and (iv) A step of selecting the antigen-binding molecules that do not bind to the first antigen in the step (iii); and (c) A step of producing a bispecific antibody comprising the variable region that binds to the first antigen selected in the step (a) and the variable region that binds to the second antigen selected in the step (b). 〔13〕The method for producing a bispecific antibody according to 〔12〕, wherein the first antigen is CD3 and the second antigen is a molecule specifically expressed in cancer tissue. 〔14〕The following steps (a) to (c): (a) A step of contacting the library according to any one of 〔1〕 to 〔7〕 with a first antigen; (b) A step of recovering the antigen-binding molecules bound to the first antigen in the step (a), and (c) A step of selecting, from the population of the antigen-binding molecules bound to the first antigen in the step (b), the antigen-binding molecules containing the variable region with enhanced binding to the first antigen A method for selecting a variable region with enhanced binding to a first antigen, which comprises 〔15〕An antigen-binding molecule comprising a variable region of an antibody that can bind to three different antigens (a first antigen, a second antigen different from the first antigen, and a fourth antigen different from the first antigen and the second antigen), but does not bind to the three antigens simultaneously. 〔16〕The antigen-binding molecule according to 〔15〕, further comprising a variable region that binds to a third antigen different from the three antigens. 〔17〕The antigen-binding molecule according to 〔15〕 or 〔16〕, wherein the variable region that does not bind to the three antigens simultaneously is a variable region that does not bind to the first antigen, the second antigen, and the fourth antigen expressed on different cells simultaneously. 〔18〕The antigen-binding molecule according to any one of 〔15〕 to 〔17〕, further comprising an Fc region of an antibody. 〔19〕The antigen-binding molecule according to 〔18〕, wherein the binding activity of the Fc region to FcγR is reduced as compared with the binding activity of the Fc region of a natural human IgG1 antibody to FcγR. 〔20〕The antigen-binding molecule according to any one of 〔15〕 to 〔19〕, wherein the variable region of the antibody that can bind to the three antigens is a variable region into which at least one amino acid modification has been introduced, and the modified amino acid is an amino acid in the CDR1, CDR2, CDR3, or FR3 region of the variable region of the antibody. 〔21〕The antigen-binding molecule according to 〔20〕, wherein the modified amino acid is an amino acid at at least one position selected from Kabat numbering 31 - 35, 50 - 65, 71 - 74, and 95 - 102 of the heavy chain variable region of the antibody, and Kabat numbering 24 - 34, 50 - 56, and 89 - 97 of the light chain variable region of the antibody. 〔22〕The antigen-binding molecule according to any one of 〔15〕 to 〔21〕, wherein any one of the first antigen, the second antigen, and the fourth antigen is a molecule specifically expressed on the surface of a T cell, and the remaining two antigens are molecules expressed on the surface of a T cell or another immune cell. 〔23〕The antigen-binding molecule according to 〔22〕, wherein the remaining two antigens are FcγR, TLR, lectin, IgA, immune checkpoint molecule, TNF superfamily molecule, TNFR superfamily molecule or NK receptor molecule. 〔24〕The antigen-binding molecule according to 〔16〕 to 〔23〕, wherein the third antigen is a molecule specifically expressed in cancer tissue. 〔25〕A pharmaceutical composition comprising the antigen-binding molecule according to any one of 〔15〕 to 〔24〕 and a pharmaceutically acceptable carrier. 〔26〕A method for producing the antigen-binding molecule according to any one of 〔15〕 to 〔24〕, the method comprising steps (i) to (iv): (i) A step of preparing a library of antigen-binding molecules in which at least one amino acid in the variable region of an antibody that binds to three different antigens (a first antigen, a second antigen different from the first antigen, and a fourth antigen different from the first antigen and the second antigen) is modified, and at least one of the amino acids in the modified variable region contains variable regions that are different from each other. (ii) A step of selecting, from the prepared library, an antigen-binding molecule containing a variable region that has binding activity to the three antigens but does not bind to the three antigens simultaneously. (iii) A step of culturing a host cell containing a nucleic acid encoding the variable region of the antigen-binding molecule selected in step (ii) to express an antigen-binding molecule containing a variable region of an antibody that can bind to the three antigens but does not bind to the three antigens simultaneously, and (iv) A step of recovering the antigen-binding molecule from the host cell culture. 〔27〕The production method according to 〔26〕, wherein the variable region that is not bound to the three antigens simultaneously, which is contained in the antigen-binding molecule selected in step (ii), is a variable region that is not bound to the three antigens expressed on different cells simultaneously. 〔28〕The production method according to 〔26〕 or 〔27〕, wherein the host cell cultured in step (iii) further contains a nucleic acid encoding the Fc region of an antibody. 〔29〕The production method according to 〔28〕, wherein the binding activity of the Fc region to FcγR is reduced as compared with the binding activity of the Fc region of a natural human IgG1 antibody to FcγR. 〔30〕The production method according to any one of 〔26〕 to 〔29〕, wherein the antigen-binding molecule to be produced is a multispecific antibody. 〔31〕The production method according to any one of 〔26〕 to 〔30〕, wherein at least one modified amino acid in the variable region in step (i) is an amino acid substituted or inserted. 〔32〕The production method according to 〔31〕, wherein the number of inserted amino acids is 1 to 25. 〔33〕The production method according to any one of 〔26〕 to 〔32〕, wherein the modification is a modification of an amino acid in the CDR1, CDR2, CDR3 or FR3 region of the variable region of the antibody. 〔34〕The production method according to any one of 〔26〕 to 〔33〕, wherein the modification is a modification of an amino acid in a loop region. 〔35〕The production method according to any one of 〔26〕 to 〔33〕, wherein the modification is a modification of at least one amino acid selected from Kabat numbering 31 to 35, 50 to 65, 71 to 74 and 95 to 102 of the heavy chain variable region of the antibody, and Kabat numbering 24 to 34, 50 to 56 and 89 to 97 of the light chain variable region. 〔36〕The production method according to any one of 〔26〕 to 〔35〕, wherein any one of the first antigen, the second antigen and the fourth antigen is a molecule specifically expressed on the surface of T cells, and the remaining two antigens are molecules expressed on the surface of T cells or other immune cells. 〔37〕The production method according to 〔36〕, wherein any one of the first antigen, the second antigen and the fourth antigen is CD3, and the remaining two antigens are FcγR, TLR, IgA, lectin, immune checkpoint molecule, TNF superfamily molecule, TNFR superfamily molecule or NK receptor molecule. 〔38〕The production method according to any one of 〔26〕 to 〔37〕, wherein the antigen-binding molecule further comprises a variable region that binds to a third antigen different from the three antigens. The production method according to

[38] , wherein the third antigen is a molecule specifically expressed in cancer tissue. 〔40〕A method for treating cancer, comprising the step of administering an antigen-binding molecule according to any one of

[15] to

[24] . 〔41〕An antigen-binding molecule according to any one of

[15] to

[24] for use in the treatment of cancer. 〔42〕Use of an antigen-binding molecule according to any one of

[15] to

[24] in the manufacture of a therapeutic agent for cancer. 〔43〕A process for manufacturing a therapeutic agent for cancer, comprising the step of using an antigen-binding molecule according to any one of

[15] to

[24] . It is understood by those skilled in the art that any combination of one or more of the above-described aspects is also included in the present invention as long as it is not technically inconsistent based on the common general knowledge of those skilled in the art.

Brief Description of the Drawings

[0016]

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Mode for Carrying Out the Invention

[0017] In one aspect, the present invention relates to a library mainly composed of a plurality of antigen-binding molecules having different sequences from each other, wherein the antigen-binding regions in the antigen-binding molecules are variable regions of antibodies (hereinafter also referred to as variable regions of antibodies of the present invention) that can bind to a first antigen and a second antigen different from the first antigen, but cannot bind to the first antigen and the second antigen simultaneously. In the antigen-binding regions contained in the library of the present invention, preferably, either one of the first antigen and the second antigen is CD3, and the other antigen is a molecule expressed on the surface of T cells or other immune cells.

[0018] In another aspect, the present invention relates to an antigen-binding molecule containing a variable region of an antibody that can bind to three different antigens (a first antigen, a second antigen, and a fourth antigen), but cannot bind to the three antigens simultaneously. Here, the "three different antigens" in the present invention refer to a first antigen, a second antigen different from the first antigen, and a fourth antigen different from the first antigen and the second antigen.

[0019] In this specification, each antigen included in the term "two different antigens" shall be represented by the terms "first antigen" and "second antigen", and each antigen included in the term "three different antigens" shall be represented by the terms "first antigen", "second antigen" and "fourth antigen".

[0020] Furthermore, the present invention relates to an antigen-binding molecule further comprising a variable region that binds to a "third antigen", which is an antigen different from the above-mentioned "three different antigens".

[0021] In the present invention, the "variable region of an antibody" generally means a region composed of four framework regions (FR) and three complementarity-determining regions (CDR) sandwiched therebetween, and as long as it has the activity of binding to part or all of an antigen, its partial sequence is also included. In particular, a region including the variable region of the antibody light chain (VL) and the variable region of the antibody heavy chain (VH) is preferred. The variable region of the antibody of the present invention may have any sequence, and may be the variable region of an antibody derived from any origin, such as a mouse antibody, a rat antibody, a rabbit antibody, a goat antibody, a camel antibody, a humanized antibody obtained by humanizing these non-human antibodies, and a human antibody. The "humanized antibody" is also referred to as a reshaped human antibody, and is obtained by transplanting the complementarity-determining region (CDR) of an antibody derived from a non-human mammal, such as a mouse antibody, into the CDR of a human antibody. Methods for identifying CDRs are known (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al., Nature (1989) 342: 877). Also, its general genetic recombination techniques are known (see European Patent Application Publication No. EP 125023, WO 96 / 02576).

[0022] When it is stated that the "variable region of the antibody" of the present invention "does not bind to the first antigen and the second antigen simultaneously", it means that when the variable region of the antibody of the present invention is bound to the first antigen, it cannot bind to the second antigen, and conversely, when the variable region is bound to the second antigen, it cannot bind to the first antigen. That is, when it is stated that the "variable region of the antibody (antibody variable region)" of the present invention "can bind to the first antigen and the second antigen, but does not bind to the first antigen and the second antigen simultaneously" or "binds to the first antigen and the second antigen and does not bind to the first antigen and the second antigen simultaneously", it means that when the variable region of the antibody of the present invention is bound to the first antigen, it cannot bind to the second antigen, and when it is not bound to the first antigen, it can bind to the second antigen. Conversely, when the variable region is bound to the second antigen, it cannot bind to the first antigen, and when it is not bound to the second antigen, it can bind to the first antigen. Here, the fact that "it does not bind to the first antigen and the second antigen simultaneously" includes not crosslinking two cells, one expressing the first antigen and the other expressing the second antigen, or not binding to the first antigen and the second antigen expressed on separate cells simultaneously. Furthermore, when the first antigen and the second antigen are not expressed on the cell membrane, such as in the case of soluble proteins, or when both are present on the same cell, it can bind to both the first antigen and the second antigen simultaneously, but when they are expressed on different cells, it also includes the case where it cannot bind simultaneously. The variable region of such an antibody is not particularly limited as long as it has such a function, and examples thereof include a variable region obtained by modifying some amino acids of the variable region of an IgG-type antibody so as to bind to a desired antigen. As the amino acids to be modified, for example, among the variable regions of antibodies that bind to the first antigen or the second antigen, amino acids that do not lose binding to the antigen due to amino acid modification are selected. Here, "expressed on different cells" means that it may be expressed on separate cells, and examples of such combinations of cells include the same type of cells such as T cells and another T cell, or different types of cells such as T cells and NK cells.

[0023] The fact that the "variable region of the antibody" of the present invention "does not bind simultaneously to three different antigens (the first antigen, the second antigen, and the fourth antigen)" means that the antigens to which the variable region of the antibody of the present invention can bind simultaneously are, among the three different antigens (i.e., three types of antigens), (A) only any one of the antigens, or (B) a combination of any two of the antigens. In other words, the fact that the variable region of the antibody of the present invention "does not bind simultaneously to three different antigens (the first antigen, the second antigen, and the fourth antigen)" means that (A) in a state where the variable region of the antibody binds to any one of the three different antigens (three types of antigens), it cannot bind to the remaining two antigens, or (B) in a state where the variable region of the antibody binds to any two of the three different antigens (three types of antigens), it cannot bind to the remaining one antigen. Specifically, the above (A) means that (i) in a state where the variable region of the antibody of the present invention binds to the first antigen, the variable region cannot bind to either the second antigen or the fourth antigen, (ii) in a state where the variable region binds to the second antigen, the variable region cannot bind to either the first antigen or the fourth antigen, and (iii) in a state where the variable region binds to the fourth antigen, the variable region cannot bind to either the first antigen or the second antigen. Specifically, the above (B) means that (i) in a state where the variable region of the antibody of the present invention binds to the first antigen and the second antigen, the variable region cannot bind to the fourth antigen, (ii) in a state where the variable region of the antibody of the present invention binds to the first antigen and the fourth antigen, the variable region cannot bind to the second antigen, and (iii) in a state where the variable region of the antibody of the present invention binds to the second antigen and the fourth antigen, the variable region cannot bind to the first antigen. The variable region of the antibody of the present invention does not bind to three different antigens simultaneously, i.e., in addition to not binding to the first antigen, the second antigen, and the fourth antigen simultaneously, it preferably does not bind to the first antigen and the second antigen simultaneously, does not bind to the first antigen and the fourth antigen simultaneously, and / or does not bind to the second antigen and the fourth antigen simultaneously. Most preferably, the variable region of the antibody of the present invention does not bind to at least two or more antigens selected from the group consisting of the first antigen, the second antigen, and the fourth antigen simultaneously (i.e., does not bind simultaneously to the combination of the first antigen and the second antigen, the combination of the first antigen and the fourth antigen, the combination of the second antigen and the fourth antigen, and the combination of the first antigen, the second antigen, and the fourth antigen). Here, "not binding to three different antigens (the first antigen, the second antigen, and the fourth antigen) simultaneously" includes not cross-linking two or more cells selected from the group consisting of cells expressing the first antigen, cells expressing the second antigen, and cells expressing the fourth antigen, or not binding simultaneously to two or more antigens selected from the group consisting of the first antigen, the second antigen, and the fourth antigen expressed on separate cells, respectively. Furthermore, if any one or more of the three different antigens (the first antigen, the second antigen, and the fourth antigen) are not expressed on the cell membrane like a soluble protein, or if any two or more of the three different antigens are present on the same cell, they can bind simultaneously to those antigens, but it also includes the case where they cannot bind simultaneously when expressed on different cells. The variable region of such an antibody is not particularly limited as long as it has such a function. For example, a variable region in which some amino acids of the variable region of an IgG-type antibody are modified to bind to a desired antigen can be mentioned.

[0024] The amino acid modifications of the present invention may be used alone or in combination of multiple sets. When used in combination, the number of combinations is not particularly limited and can be appropriately set within the range that can achieve the object of the invention. For example, it can be 2 or more and 30 or less, preferably 2 or more and 25 or less, 2 or more and 22 or less, 2 or more and 20 or less, 2 or more and 15 or less, 2 or more and 10 or less, 2 or more and 5 or less, 2 or more and 3 or less. When combining multiple, the amino acid modification may be added only to the heavy chain variable region or the light chain variable region of the antibody, or may be appropriately distributed and added to both the heavy chain variable region and the light chain variable region.

[0025] As long as the antigen-binding activity is maintained, modification of one or more amino acid residues in the variable region is allowed for the amino acid residue to be modified (modified amino acid). When modifying the amino acid in the variable region, although not particularly limited, it is preferable that the binding activity of the antibody before modification is maintained. For example, it preferably has a binding activity of 50% or more, preferably 80% or more, more preferably 100% or more compared to before modification. Also, the binding activity may be increased by amino acid modification. For example, the binding activity may be 2-fold, 5-fold, 10-fold, etc. compared to before modification.

[0026] Preferred regions for amino acid modification include regions exposed to the solvent and loop regions in the variable region. Among them, CDR1, CDR2, CDR3, FR3 regions, and loop regions are preferred. Specifically, Kabat numbering 31 - 35, 50 - 65, 71 - 74, 95 - 102 of the H chain variable region and Kabat numbering 24 - 34, 50 - 56, 89 - 97 of the L chain variable region are preferred, and Kabat numbering 31, 52a - 61, 71 - 74, 97 - 101 of the H chain variable region and Kabat numbering 24 - 34, 51 - 56, 89 - 96 of the L chain variable region are more preferred. Also, when modifying amino acids, amino acids that increase the binding activity with the antigen may be introduced together.

[0027] In the present invention, the "loop region" means a region where residues that do not participate in maintaining the β-barrel structure of the immunoglobulin exist. In the present invention, the modification of an amino acid means any of substitution, deletion, addition, insertion, or modification, or a combination thereof. In the present invention, the modification of an amino acid can be rephrased as a mutation of an amino acid and is used in the same meaning.

[0028] When substituting an amino acid residue, the purpose is to modify the following points (a) to (c) by substituting it with another amino acid residue. (a) The backbone structure of the polypeptide in the region of the sheet structure or the helical structure; (b) The charge or hydrophobicity at the target site, or (c) The size of the side chain. Amino acid residues are classified into the following groups based on the characteristics of general side chains: (1) Hydrophobic: norleucine, met, ala, val, leu, ile; (2) Neutral hydrophilic: cys, ser, thr, asn, gln; (3) Acidic: asp, glu; (4) Basic: his, lys, arg; (5) Residues that affect the orientation of the chain: gly, pro; and (6) Aromatic: trp, tyr, phe.

[0029] The substitution of amino acid residues within each of these groups is called a conservative substitution, while the substitution of amino acid residues between different groups is called a non-conservative substitution. The substitution in the present invention may be a conservative substitution, a non-conservative substitution, or a combination of a conservative substitution and a non-conservative substitution.

[0030] In addition, when modifying amino acid residues, among those obtained by randomly modifying amino acids in the variable region of an antibody that binds to the first antigen or the second antigen and that do not cause loss of binding to the antigen due to the amino acid modification, a variable region that can bind to the first antigen and the second antigen but cannot bind to these antigens simultaneously is selected, or modification including inserting a peptide known in advance to have binding activity to a desired antigen into the above region is also included. Further, among those obtained by randomly modifying amino acids in the variable region of an antibody that binds to the first antigen, the second antigen, or the fourth antigen and that do not cause loss of binding to the antigen due to the amino acid modification, a variable region that can bind to the first antigen, the second antigen, and the fourth antigen but cannot bind to these antigens simultaneously is selected, or modification including inserting a peptide known in advance to have binding activity to a desired antigen into the above region is also included. Examples of the peptide known in advance to have binding activity to a desired antigen include the peptides shown in Table 1.

[0031]

Table 1

[0032] As one aspect of the present invention, there is provided an antigen-binding molecule comprising a variable region of an antibody in which the amino acids of the heavy-chain variable region are modified so as to be able to bind to a first antigen and a second antigen different from the first antigen, but not to bind to the first antigen and the second antigen simultaneously. As another aspect of the present invention, there is provided an antigen-binding molecule comprising a variable region of an antibody in which the amino acids of the heavy-chain variable region are modified so as to be able to bind to three different antigens (a first antigen, a second antigen, and a fourth antigen), but not to bind to the three antigens simultaneously. For example, by introducing the above-described amino acid modifications (any one of substitution, deletion, addition, insertion, or modification, or a combination thereof) into the heavy-chain variable region, it is possible to obtain a variable region of an antibody that can bind to a first antigen and a second antigen different from the first antigen, but does not bind to the first antigen and the second antigen simultaneously, or a variable region of an antibody that can bind to three different antigens (a first antigen, a second antigen, and a fourth antigen), but does not bind to the three antigens simultaneously. The position where the amino acid modification is introduced is preferably the heavy-chain variable region, and more preferably regions exposed to the solvent and loop regions in the variable region. Among them, CDR1, CDR2, CDR3, FR3 regions, and loop regions are preferred. Specifically, Kabat numbering 31-35, 50-65, 71-74, 95-102 of the H-chain variable region is preferred, and Kabat numbering 31, 52a-61, 71-74, 97-101 of the H-chain variable region is more preferred. Further, when introducing amino acid modifications, amino acids that increase the binding activity to the antigen may be introduced together.

[0033] In addition to the above-described modifications, known modifications may be combined with the variable region of the antibody of the present invention. For example, modification to pyroglutamic acid by pyroglutamylation of glutamine at the N-terminus of the variable region is a modification well known to those skilled in the art. Therefore, the antibody of the present invention, when the N-terminus of its heavy chain is glutamine, includes a variable region in which it is modified to pyroglutamic acid.

[0034] In addition, for example, amino acid modifications may be further introduced into the variable regions of these antibodies to improve antigen binding, pharmacokinetics, stability, and antigenicity. By adding a modification to the variable region of the antibody of the present invention to have pH-dependent binding to the antigen, it may be possible to repeatedly bind to the antigen (WO / 2009 / 125825).

[0035] In addition, for example, amino acid modifications can also be added to the variable regions that bind to the third antigen of these antibodies, such that the binding activity to the antigen changes according to the concentration of the target tissue-specific compound (WO2013 / 180200).

[0036] Furthermore, for example, modifications of the variable region are identified using in silico prediction or by assays using in vitro T cells for the purpose of increasing binding activity, improving specificity, decreasing pI, imparting pH-dependent properties to antigen binding, improving binding thermal stability, improving solubility, stability against chemical modification, improving heterogeneity derived from sugar chains, avoiding T cell epitopes identified by in vitro T cell assays, or introducing T cell epitopes that activate regulatory T cells (mAbs 3:243-247, 2011). Modifications for such purposes can also be carried out.

[0037] Whether the variable region of the antibody of the present invention "can bind to the first antigen and the second antigen" can be measured using known methods. For example, it can be measured by the electrochemiluminescence method (ECL method) (BMC Research Notes 2011, 4:281). Specifically, for example, a region capable of binding to a first antigen and a second antigen of a biotin-labeled test antigen-binding molecule, such as a Fab region, a low-molecular-weight antibody composed thereof, or a monovalent antibody (an antibody lacking one of the two Fab regions of a normal antibody), is mixed with a first antigen or a second antigen labeled with a sulfo-tag (Ru complex) and added onto a streptavidin-immobilized plate. At this time, the biotin-labeled test antigen-binding molecule binds to streptavidin on the plate. By emitting light from the sulfo-tag and detecting the emitted light signal using a Sector Imager 600, 2400 (MSD), etc., it is possible to confirm the binding between the first antigen or the second antigen and the above-mentioned region of the test antigen-binding molecule. It can also be measured by ELISA, FACS (fluorescence activated cell sorting), ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), BIACORE method using surface plasmon resonance (SPR) phenomenon, etc. (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005 - 4010).

[0038] Specifically, for example, it can be measured using Biacore (GE Healthcare), which is an interaction analysis instrument utilizing the surface plasmon resonance (SPR) phenomenon. Biacore includes any model such as Biacore T100, T200, X100, A100, 4000, 3000, 2000, 1000, C, etc. Any of the sensor chips for Biacore, such as CM7, CM5, CM4, CM3, C1, SA, NTA, L1, HPA, Au chip, etc., can be used. Proteins for capture, such as ProteinA, ProteinG, ProteinL, anti-human IgG antibody, anti-human IgG-Fab, anti-human light chain antibody, anti-human Fc antibody, antigen protein, antigen peptide, etc., are immobilized on the sensor chip by coupling methods such as amine coupling, disulfide coupling, aldehyde coupling, etc., to capture the antigen-binding molecule of the present invention. Then, the first antigen or the second antigen is flowed as an analyte to measure the interaction and obtain a sensorgram. At this time, the concentration of the first antigen or the second antigen can be carried out in the range of several μM to several pM according to the strength of the interaction such as the KD of the sample to be measured.

[0039] Alternatively, instead of the antigen-binding molecule, it is also possible to immobilize the first antigen or the second antigen on the sensor chip and allow the antibody sample to be evaluated to interact therewith. It is possible to determine whether the antibody variable region of the antigen-binding molecule of the present invention has binding activity to the first antigen or the second antigen from the dissociation constant (KD) value calculated from the sensorgram of the interaction or from the degree of increase in the sensorgram before and after the action of the antigen-binding molecule sample.

[0040] The AlphaScreen is implemented based on the following principle by the Alpha technology that uses two beads, a donor bead and an acceptor bead. When the molecule bound to the donor bead biologically interacts with the molecule bound to the acceptor bead, a luminescence signal is detected only when the two beads are in proximity. The photosensitizer in the donor bead excited by the laser converts the surrounding oxygen into singlet oxygen in the excited state. The singlet oxygen diffuses around the donor bead and, when it reaches the nearby acceptor bead, causes a chemiluminescence reaction in the bead, and finally light is emitted. When the molecule bound to the donor bead and the molecule bound to the acceptor bead do not interact, the singlet oxygen produced by the donor bead does not reach the acceptor bead, so the chemiluminescence reaction does not occur.

[0041] One of the substances to observe the interaction (ligand) is immobilized on the gold thin film of the sensor chip. When light is applied from the back side of the sensor chip so as to totally reflect at the interface between the gold thin film and the glass, a part of the reflected light with a reduced reflection intensity (SPR signal) is formed. When the other substance to observe the interaction (analyte) is flowed onto the surface of the sensor chip and the ligand and the analyte bind to each other, the mass of the immobilized ligand molecules increases, and the refractive index of the solvent on the surface of the sensor chip changes. Due to this change in the refractive index, the position of the SPR signal shifts (conversely, when the binding dissociates, the position of the signal returns). The Biacore system takes the amount of the above shift, that is, the mass change on the surface of the sensor chip, on the vertical axis, and displays the time change of the mass as measurement data (sensorgram). From the sensorgram, the amount of binding of the analyte to the ligand captured on the surface of the sensor chip (the amount of change in the response on the sensorgram before and after the analyte is allowed to interact) is determined. However, since the amount of binding also depends on the amount of the ligand, it is necessary to compare under conditions where the amount of the ligand can be regarded as being essentially the same amount when comparing. Also, from the curve of the sensorgram, the kinetics: the association rate constant (ka) and the dissociation rate constant (kd) are obtained, and the affinity (KD) is obtained from the ratio of the constants. The inhibition measurement method is also preferably used in the BIACORE method. An example of the inhibition measurement method is described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

[0042] Whether the variable region of the antibody of the present invention "can bind to three different antigens (the first antigen, the second antigen, and the fourth antigen)" can be appropriately measured by those skilled in the art using, for example, the electrochemiluminescence (ECL) method or the like according to the known methods described above.

[0043] Whether the antigen-binding molecule of the present invention "does not bind to the first antigen and the second antigen simultaneously" can be confirmed by, after confirming that it has binding activity to the first antigen and the second antigen, using the above method to measure whether the antigen-binding molecule containing the variable region having the binding activity has binding activity to the remaining one after binding either the first antigen or the second antigen in advance. It can also be confirmed by measuring whether the binding of the antigen-binding molecule to either the first antigen or the second antigen immobilized on an ELISA plate or a sensor chip is inhibited by adding the other in solution.

[0044] Specifically, for example, when using the ECL method, prepare a biotin-labeled test antigen-binding molecule, a first antigen labeled with sulfo-tag (Ru complex), and an unlabeled second antigen. If the test antigen-binding molecule can bind to the first antigen and the second antigen but does not bind to the first antigen and the second antigen simultaneously, in the absence of the unlabeled second antigen, when a mixture of the test antigen-binding molecule and the first antigen is added onto a streptavidin-immobilized plate and the sulfo-tag is made to emit light, the emission signal is detected, but in the presence of the second antigen, the emission signal decreases. The relative binding activity can be determined by quantifying the decrease in this emission signal.

[0045] Also, in the case of AlphaScreen, in the absence of a competing second antigen, the test antigen-binding molecule and the first antigen interact to produce a signal at 520 - 620 nm. The untagged second antigen competes with the interaction between the test antigen-binding molecule and the first antigen. The relative binding activity can be determined by quantifying the resulting decrease in fluorescence. Biotinylation of polypeptides using, for example, Sulfo-NHS-biotin is known. As a method for tagging the first antigen with GST, a fusion gene in which a polynucleotide encoding the first antigen and a polynucleotide encoding GST are fused in-frame is expressed in cells or the like holding a vector capable of expressing the fusion gene, and purification is carried out using a glutathione column or the like, and such methods can be appropriately employed. The obtained signal is preferably analyzed by fitting it to a one-site competition model using non-linear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego). At this time, it is also possible to carry out the analysis in the same manner by tagging the second antigen and not tagging the first antigen. In addition, a method using fluorescence resonance energy transfer (FRET) can also be employed. FRET is a phenomenon in which excitation energy directly moves between two adjacent fluorescent molecules by electron resonance. When FRET occurs, the excitation energy of the donor (a fluorescent molecule in the excited state) moves to the acceptor (another fluorescent molecule in the vicinity of the donor), so the fluorescence emitted from the donor disappears (more precisely, the fluorescence lifetime is shortened), and instead, fluorescence is emitted from the acceptor. This phenomenon can be used to analyze whether it is a dual-Fab. For example, when a first antigen introduced with a fluorescent donor and a second antigen introduced with a fluorescent acceptor bind to the test antigen-binding molecule simultaneously, the fluorescence of the donor disappears and fluorescence is emitted from the acceptor, resulting in a change in the fluorescence wavelength. Such an antibody is judged not to be a dual-Fab. On the other hand, when the first antigen, the second antigen, and the test antigen-binding molecule are mixed, if there is no change in the fluorescence wavelength of the fluorescent donor bound to the first antigen, it can be said that this test antigen-binding molecule is a dual-Fab.

[0046] For example, a test antigen-binding molecule labeled with biotin binds to streptavidin on donor beads, and a first antigen tagged with glutathione S-transferase (GST) binds to acceptor beads. In the absence of a competing second antigen, the test antigen-binding molecule and the first antigen interact to produce a signal at 520-620 nm. An untagged second antigen competes with the interaction between the test antigen-binding molecule and the first antigen. The relative binding activity can be determined by quantifying the resulting decrease in fluorescence. Biotinylation of a polypeptide using, for example, Sulfo-NHS-biotin is known. As a method for tagging the first antigen with GST, a fusion gene in which a polynucleotide encoding the first antigen and a polynucleotide encoding GST are fused in-frame is expressed in cells or the like that hold an expressible vector, and a method of purifying using a glutathione column or the like can be appropriately employed. The obtained signal is preferably analyzed by fitting it to a one-site competition model using non-linear regression analysis with software such as GRAPHPAD PRISM (GraphPad, San Diego).

[0047] Note that the tagging is not limited to GST and can be any tag such as a histidine tag, MBP, CBP, Flag tag, HA tag, V5 tag, c-myc tag, etc., and is not limited. Also, regarding the binding of the test antigen-binding molecule to the donor beads, it is limited to binding using the biotin-streptavidin reaction. In particular, when the test antigen-binding molecule contains Fc, a method of binding the test antigen-binding molecule via an Fc-recognizing protein such as Protein A or Protein G on the donor beads can be considered.

[0048] Also, when the first antigen and the second antigen are not expressed on the cell membrane like a soluble protein, or when both are present on the same cell, they can bind to both the first antigen and the second antigen simultaneously. However, when they are expressed on different cells, it is also possible to measure using a known method even when they cannot bind simultaneously. Specifically, even when positive in an ECL-ELISA for detecting simultaneous binding to a first antigen and a second antigen, if the three components, namely the cells expressing the first antigen, the cells expressing the second antigen, and the test antigen-binding molecule, do not bind simultaneously when mixed, it can be shown that they cannot bind simultaneously when expressed on different cells. For example, it can be measured by the ECL-ELISA method using cells. First, cells expressing the first antigen are immobilized on a plate, the test antigen-binding molecule is bound, and then cells expressing the second antigen are added. By detecting with a sulfo-tag-labeled antibody against another antigen expressed only on the cells expressing the second antigen, a signal is observed when the two antigens expressed on the two cells bind simultaneously, and no signal is observed when they do not bind simultaneously. Alternatively, it can be measured by the alphascreen method. When cells expressing the first antigen bound to donor beads, cells expressing the second antigen bound to acceptor beads, and the test antigen-binding molecule are mixed, a signal is observed when the two antigens expressed on the two cells bind simultaneously, and no signal is observed when they do not bind simultaneously. Alternatively, it can be measured by an interaction analysis method using Octet. First, cells expressing the first antigen with a peptide tag added are bound to a biosensor that recognizes the peptide tag. When an interaction analysis is performed in a well containing cells expressing the second antigen and the test antigen-binding molecule, a large wavelength shift is observed when the two antigens expressed on the two cells bind simultaneously because the test antigen-binding molecule and the cells expressing the second antigen bind to the biosensor, and a small wavelength shift is observed when they do not bind simultaneously because only the test antigen-binding molecule binds to the biosensor.

[0049] Alternatively, it is also possible to measure by biological activity instead of binding activity. For example, when cells expressing a first antigen, cells expressing a second antigen, and a test antigen-binding molecule are co-cultured, if they are simultaneously bound to the two antigens expressed on the two cells, they are mutually activated via the test antigen-binding molecule, so changes in activation signals such as an increase in phosphorylation downstream of each antigen can be detected. Or, as a result of activation, cytokine production is induced, so by measuring the amount of cytokine produced, it is possible to determine whether or not it binds to the two cells simultaneously.

[0050] Whether or not the antigen-binding molecule of the present invention "does not bind to three different antigens simultaneously" can be appropriately measured by those skilled in the art using, for example, the electrochemiluminescence (ECL) method or the like according to the known methods described above after confirming that it has binding activity against three different antigens (the first antigen, the second antigen, and the fourth antigen).

[0051] In the present invention, the "Fc region" refers to a region in an antibody molecule that includes a fragment consisting of a hinge portion or a part thereof, CH2, and CH3 domains. The Fc region of the IgG class, in the EU numbering (also referred to as EU INDEX in this specification), means, for example, from the 226th cysteine to the C-terminus, or from the 230th proline to the C-terminus, but is not limited thereto. The Fc region can be preferably obtained by partially digesting IgG1, IgG2, IgG3, IgG4 monoclonal antibodies or the like with a proteolytic enzyme such as pepsin and then redissolving the fraction adsorbed to a protein A column or a protein G column. Such a proteolytic enzyme is not particularly limited as long as it can digest the full-length antibody to restrictively produce Fab or F(ab')2 by appropriately setting the reaction conditions of the enzyme such as pH, and examples thereof include pepsin and papain.

[0052] The "antigen-binding molecule" in the present invention is not particularly limited as long as it is a molecule containing the "variable region of an antibody" of the present invention, and may further contain a peptide or protein having a length of about 5 amino acids or more. It is not limited to peptides and proteins derived from organisms, and may be, for example, a polypeptide consisting of an artificially designed sequence. It may be any of natural polypeptides, synthetic polypeptides, recombinant polypeptides, etc.

[0053] Preferred examples of the antigen-binding molecule of the present invention include antigen-binding molecules containing the Fc region of an antibody.

[0054] As the "Fc region" of the present invention, for example, an Fc region derived from natural IgG can be used. Here, natural IgG means a polypeptide that includes the same amino acid sequence as that found in nature and belongs to the class of antibodies substantially encoded by the immunoglobulin gamma gene. For example, natural human IgG means natural human IgG1, natural human IgG2, natural human IgG3, natural human IgG4, etc. Natural IgG also includes naturally occurring variants and the like. Multiple allotype sequences due to genetic polymorphisms are described in Sequences of proteins of immunological interest, NIH Publication No.91-3242 for the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, and any of them may be used in the present invention. In particular, for the sequence of human IgG1, the amino acid sequences at positions 356-358 in EU numbering may be DEL or EEM.

[0055] As the Fc region of the antibody, for example, there are Fc regions of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, IgM types. As the Fc region of the antibody of the present invention, for example, an Fc region derived from a native human IgG antibody can be used. As the Fc region of the present invention, for example, a constant region of native IgG, specifically, a constant region derived from native human IgG1 (SEQ ID NO: 1), a constant region derived from native human IgG2 (SEQ ID NO: 2), a constant region derived from native human IgG3 (SEQ ID NO: 3), a constant region derived from native human IgG4 (SEQ ID NO: 4) can be used. The constant region of native IgG also includes naturally occurring variants and the like.

[0056] As the Fc region of the present invention, an Fc region with particularly reduced binding activity to the Fcγ receptor is preferable. Here, the Fcγ receptor (which may be described as Fcγ receptor, FcγR or Fcγ receptor in this specification) refers to a receptor that can bind to the Fc regions of IgG1, IgG2, IgG3, and IgG4, and substantially means any member of the family of proteins encoded by the Fcγ receptor gene. In humans, this family includes FcγRI (CD64) including isoforms FcγRIa, FcγRIb and FcγRIc; FcγRII (CD32) including isoforms FcγRIIa (including allotypes H131 (H type) and R131 (R type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) and FcγRIIc; and FcγRIII (CD16) including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), and also includes any undiscovered human FcγRs or FcγR isoforms or allotypes, but is not limited thereto. FcγRs include those derived from humans, mice, rats, rabbits and monkeys, but are not limited thereto and may be derived from any organism. Mouse FcγRs include FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16) and FcγRIII-2 (CD16-2), and also include any undiscovered mouse FcγRs or FcγR isoforms or allotypes, but are not limited thereto. Preferred examples of such Fcγ receptors include human FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16) and / or FcγRIIIb (CD16).

[0057] FcγRs include activating receptors with ITAM (Immunoreceptor tyrosine - based activation motif) and inhibitory receptors with ITIM (immunoreceptor tyrosine - based inhibitory motif). FcγRs are classified into activating FcγRs such as FcγRI, FcγRIIa R, FcγRIIa H, FcγRIIIa, FcγRIIIb and inhibitory FcγR of FcγRIIb. The polynucleotide sequences and amino acid sequences of FcγRI are described in NM_000566.3 and NP_000557.1 respectively, those of FcγRIIa are described in BC020823.1 and AAH20823.1 respectively, those of FcγRIIb are described in BC146678.1 and AAI46679.1 respectively, those of FcγRIIIa are described in BC033678.1 and AAH33678.1 respectively, and those of FcγRIIIb are described in BC128562.1 and AAI28563.1 respectively (RefSeq accession numbers). Note that for FcγRIIa, there are two genetic polymorphisms in which the 131st amino acid of FcγRIIa is substituted with histidine (H type) or arginine (R type) (J. Exp. Med, 172, 19 - 25, 1990). Also, for FcγRIIb, there are two genetic polymorphisms in which the 232nd amino acid of FcγRIIb is substituted with isoleucine (I type) or threonine (T type) (Arthritis. Rheum. 46: 1242 - 1254 (2002)). Also, for FcγRIIIa, there are two genetic polymorphisms in which the 158th amino acid of FcγRIIIa is substituted with valine (V type) or phenylalanine (F type) (J. Clin. Invest. 100(5): 1059 - 1070 (1997)). Also, for FcγRIIIb, there are two genetic polymorphisms of NA1 type and NA2 type (J. Clin. Invest. 85: 1287 - 1295 (1990)).

[0058] Whether the binding activity to the Fcγ receptor is decreased can be confirmed by well-known methods such as FACS, ELISA format, AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay), and the BIACORE method using the surface plasmon resonance (SPR) phenomenon (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010). AlphaScreen is carried out based on the following principle by the Alpha technology using two beads, a donor bead and an acceptor bead. When a molecule bound to the donor bead biologically interacts with a molecule bound to the acceptor bead, a luminescence signal is detected only when the two beads are in proximity. The photosensitizer in the donor bead excited by a laser converts the surrounding oxygen into singlet oxygen in an excited state. The singlet oxygen diffuses around the donor bead and, when it reaches the nearby acceptor bead, causes a chemiluminescence reaction in the bead, and finally light is emitted. When the molecule bound to the donor bead and the molecule bound to the acceptor bead do not interact, the singlet oxygen produced by the donor bead does not reach the acceptor bead, so the chemiluminescence reaction does not occur.

[0059] For example, an antigen-binding molecule labeled with biotin is bound to donor beads, and an Fcγ receptor tagged with glutathione S-transferase (GST) is bound to acceptor beads. In the absence of an antigen-binding molecule having a competing mutant Fc region, the antigen-binding molecule having a wild-type Fc region and the Fcγ receptor interact to produce a signal at 520-620 nm. An antigen-binding molecule having an untagged mutant Fc region competes with the interaction between the antigen-binding molecule having a wild-type Fc region and the Fcγ receptor. The relative binding affinity can be determined by quantifying the decrease in fluorescence resulting from the competition. Biotinylation of an antigen-binding molecule such as an antibody using, for example, Sulfo-NHS-biotin is known. As a method for tagging an Fcγ receptor with GST, a fusion gene in which a polynucleotide encoding the Fcγ receptor and a polynucleotide encoding GST are fused in-frame is expressed in cells or the like that hold a vector capable of expressing the fusion gene, and a method of purifying using a glutathione column or the like can be appropriately employed. The obtained signal is preferably analyzed by fitting it to a one-site competition model using non-linear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

[0060] One of the substances to observe the interaction (ligand) is immobilized on the gold thin film of the sensor chip. When light is applied from the back side of the sensor chip so as to totally reflect at the interface between the gold thin film and the glass, a part of the reflected light with a reduced reflection intensity (SPR signal) is formed. When the other substance to observe the interaction (analyte) is flowed on the surface of the sensor chip and the ligand and the analyte bind to each other, the mass of the immobilized ligand molecules increases, and the refractive index of the solvent on the sensor chip surface changes. Due to this change in the refractive index, the position of the SPR signal shifts (conversely, when the binding dissociates, the position of the signal returns). The Biacore system takes the amount of the above shift, that is, the mass change on the sensor chip surface, on the vertical axis and displays the time change of the mass as measurement data (sensorgram). From the curve of the sensorgram, the kinetics: the association rate constant (ka) and the dissociation rate constant (kd) are obtained, and the affinity (KD) is obtained from the ratio of the constants. The inhibition measurement method is also preferably used in the BIACORE method. Examples of the inhibition measurement method are described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

[0061] In the present specification, the decrease in the binding activity to the Fcγ receptor means that, for example, based on the above analysis method, the binding activity of the test antigen-binding molecule is 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, 15% or less, particularly preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, as compared with the binding activity of the antigen-binding molecule containing the control Fc region. As the antigen-binding molecule for comparison, an antigen-binding molecule having the Fc region of an IgG1, IgG2, IgG3 or IgG4 monoclonal antibody can be appropriately used. The structure of the Fc region is described in SEQ ID NO: 1 (A added to the N-terminus of RefSeq accession number AAC82527.1), SEQ ID NO: 2 (A added to the N-terminus of RefSeq accession number AAB59393.1), SEQ ID NO: 3 (A added to the N-terminus of RefSeq accession number CAA27268.1), SEQ ID NO: 4 (A added to the N-terminus of RefSeq accession number AAB59394.1). When an antigen-binding molecule having a variant of the Fc region of an antibody of a specific isotype is used as a test substance, by using an antigen-binding molecule having the Fc region of an antibody of the specific isotype as a control, the effect on the binding activity to the Fcγ receptor due to the mutation of the variant is verified. As described above, an antigen-binding molecule having a variant of the Fc region whose binding activity to the Fcγ receptor has been verified to be decreased is appropriately prepared.

[0062] Examples of such variants include deletions of 231A-238S, which are amino acids specified according to EU numbering (WO 2009 / 011941), C226S, C229S, P238S, (C220S) (J.Rheumatol (2007) 34, 11), C226S, C229S (Hum.Antibod.Hybridomas (1990) 1(1), 47-54), C226S, C229S, E233P, L234V, L235A (Blood (2007) 109, 1185-1192), etc. are known variants. That is, among the amino acids constituting the Fc region of an antibody of a specific isotype, any of the following amino acids specified according to EU numbering; amino acids at positions 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331, 332 are substituted, and an antigen-binding molecule having an Fc region is preferably mentioned. The isotype of the antibody that is the origin of the Fc region is not particularly limited, and an Fc region derived from an IgG1, IgG2, IgG3 or IgG4 monoclonal antibody can be appropriately used, but an Fc region derived from a native human IgG1 antibody is preferably used. For example, among the amino acids constituting the Fc region of an IgG1 antibody, any of the following substitutions specified according to EU numbering (the number is the position of the amino acid residue specified according to EU numbering, the single-letter amino acid symbol located before the number represents the amino acid residue before substitution, and the single-letter amino acid symbol located after the number represents the amino acid residue before substitution); (a) L234F, L235E, P331S, (b) C226S, C229S, P238S, (c) C226S, C229S, (d) C226S, C229S, E233P, L234V, L235A An antigen-binding molecule having an Fc region subjected to the above, or an Fc region lacking the amino acid sequence from positions 231 to 238 can also be appropriately used.

[0063] In addition, among the amino acids constituting the Fc region of an IgG2 antibody, any of the following substitutions specified according to EU numbering (the number is the position of the amino acid residue specified according to EU numbering, the single-letter amino acid symbol located before the number represents the amino acid residue before substitution, and the single-letter amino acid symbol located after the number represents the amino acid residue before substitution); (e) H268Q, V309L, A330S, P331S (f) V234A (g) G237A (h) V234A, G237A (i) A235E, G237A (j) V234A, A235E, G237A Antigen-binding molecules having an Fc region in which such substitution has been made can also be appropriately used.

[0064] In addition, among the amino acids constituting the Fc region of an IgG3 antibody, any of the following substitutions specified according to EU numbering (the number represents the position of the amino acid residue specified according to EU numbering, the single-letter amino acid symbol preceding the number represents the amino acid residue before substitution, and the single-letter amino acid symbol following the number represents the amino acid residue before substitution); (k) F241A (l) D265A (m) V264A Antigen-binding molecules having an Fc region in which such substitution has been made can also be appropriately used.

[0065] In addition, among the amino acids constituting the Fc region of an IgG4 antibody, any of the following substitutions specified according to EU numbering (the number represents the position of the amino acid residue specified according to EU numbering, the single-letter amino acid symbol preceding the number represents the amino acid residue before substitution, and the single-letter amino acid symbol following the number represents the amino acid residue before substitution); (n) L235A, G237A, E318A (o) L235E (p) F234A, L235A Antigen-binding molecules having an Fc region in which such substitution has been made can also be appropriately used.

[0066] As other preferred examples, among the amino acids constituting the Fc region of a natural human IgG1 antibody, any of the following amino acids specified according to EU numbering; positions 233, 234, 235, 236, 237, 327, 330, 331, are substituted with the corresponding amino acids in the corresponding IgG2 or IgG4 according to its EU numbering, and antigen-binding molecules having an Fc region are included.

[0067] As other preferred examples, among the amino acids constituting the Fc region of a native human IgG1 antibody, any one or more of the following amino acids specified according to EU numbering; an antigen-binding molecule having an Fc region in which the amino acids at positions 234, 235, and 297 are substituted with other amino acids is preferably mentioned. The type of amino acid present after substitution is not particularly limited, but an antigen-binding molecule having an Fc region in which any one or more of the amino acids at positions 234, 235, and 297 are substituted with alanine is particularly preferred.

[0068] As other preferred examples, among the amino acids constituting the Fc region of an IgG1 antibody, any of the following amino acids specified according to EU numbering; an antigen-binding molecule having an Fc region in which the amino acid at position 265 is substituted with other amino acids is preferably mentioned. The type of amino acid present after substitution is not particularly limited, but an antigen-binding molecule having an Fc region in which the amino acid at position 265 is substituted with alanine is particularly preferred.

[0069] In addition, as one of the preferred embodiments of the "antigen-binding molecule" of the present invention, a multispecific antibody containing the variable region of the antibody of the present invention can be mentioned.

[0070] For the association of multispecific antibodies, a technique of introducing a charge repulsion at the interface of the second constant region (CH2) or the third constant region (CH3) of the antibody heavy chain to suppress the association of unwanted heavy chains can be applied (WO2006 / 106905). In the technique of introducing a charge repulsion at the interface of CH2 or CH3 to suppress the association of unintended heavy chains, examples of the amino acid residues that contact at the interface of other constant regions of the heavy chain include regions corresponding to the residue at position 356 of EU numbering, the residue at position 439 of EU numbering, the residue at position 357 of EU numbering, the residue at position 370 of EU numbering, the residue at position 399 of EU numbering, and the residue at position 409 of EU numbering in the CH3 region.

[0071] More specifically, for example, in an antibody containing two H-chain CH3 regions, one to three sets of amino acid residues selected from the following sets (1) to (3) in the first H-chain CH3 region can be an antibody in which the amino acid residues have the same charge; (1) Amino acid residues contained in the H-chain CH3 region, which are the amino acid residues at positions 356 and 439 in EU numbering, (2) Amino acid residues contained in the H-chain CH3 region, which are the amino acid residues at positions 357 and 370 in EU numbering, (3) Amino acid residues contained in the H-chain CH3 region, which are the amino acid residues at positions 399 and 409 in EU numbering.

[0072] Furthermore, a set of amino acid residues selected from the sets of amino acid residues shown in (1) to (3) above in a second H-chain CH3 region different from the first H-chain CH3 region, and one to three sets of amino acid residues corresponding to the sets of amino acid residues shown in (1) to (3) above that have the same charge in the first H-chain CH3 region have a charge opposite to that of the corresponding amino acid residues in the first H-chain CH3 region can be an antibody.

[0073] Each of the amino acid residues described in (1) to (3) above is close to each other when associated. A person skilled in the art can find the sites corresponding to the amino acid residues described in (1) to (3) above by homology modeling or the like using commercially available software for a desired H-chain CH3 region or H-chain constant region, and can appropriately subject the amino acid residues at the sites to modification.

[0074] In the above antibody, the "amino acid residue having a charge" is preferably selected from amino acid residues contained in any one of the following groups (a) or (b); (a) Glutamic acid (E), aspartic acid (D), (b) Lysine (K), arginine (R), histidine (H).

[0075] In the above antibody, "having the same charge" means, for example, that any of two or more amino acid residues has an amino acid residue included in any one of the groups (a) or (b) above. "Having opposite charges" means, for example, that when at least one of two or more amino acid residues has an amino acid residue included in any one of the groups (a) or (b) above, the remaining amino acid residues have amino acid residues included in a different group.

[0076] In a preferred embodiment, in the above antibody, the first H-chain CH3 region and the second H-chain CH3 region may be cross-linked by a disulfide bond. The amino acid residues to be modified in the present invention are not limited to the amino acid residues in the variable region or the constant region of the antibody described above. Those skilled in the art can find amino acid residues forming an interface by homology modeling or the like using commercially available software for polypeptide variants or heteromultimers, and can subject the amino acid residues at the site to modification so as to control the association.

[0077] In addition, other known techniques can also be used for the association of the multispecific antibody of the present invention. By substituting the amino acid side chain present in the variable region of one H-chain of the antibody with a larger side chain (knob; protrusion) and substituting the amino acid side chain present in the opposing variable region of the other H-chain with a smaller side chain (hole; cavity), the protrusion can be arranged in the cavity, thereby efficiently causing the association of polypeptides having different amino acids having an Fc region (WO1996 / 027011, Ridgway JB et al., Protein Engineering (1996) 9, 617-621, Merchant AM et al. Nature Biotechnology (1998) 16, 677-681).

[0078] In addition to this, other known techniques can also be used to form the multispecific antibodies of the present invention. By using strand-exchange engineered domain CH3, in which a part of CH3 of one H chain of the antibody is replaced with a sequence derived from IgA corresponding to that part and a sequence derived from IgA corresponding to that part is introduced into the complementary part of CH3 of the other H chain, the association of polypeptides having different sequences can be efficiently induced by the complementary association of CH3 (Protein Engineering Design & Selection, 23; 195-202, 2010). Using this known technique, the desired multispecific antibodies can also be efficiently formed.

[0079] For the formation of multispecific antibodies, other techniques can also be used, such as the antibody production technique that utilizes the association of CH1 and CL of antibodies described in WO2011 / 028952, the association of VH and VL, the technique of producing bispecific antibodies using separately prepared monoclonal antibodies described in WO2008 / 119353 and WO2011 / 131746 (Fab Arm Exchange), the technique of controlling the association between CH3 of antibody heavy chains described in WO2012 / 058768 and WO2013 / 063702, the technique of producing bispecific antibodies composed of two types of light chains and one type of heavy chain described in WO2012 / 023053, the technique of producing bispecific antibodies using two bacterial cell lines that express single chains of antibodies consisting of one H chain and one L chain described in Christoph et al. (Nature Biotechnology Vol. 31, p 753-758 (2013)), etc. In addition to the above association techniques, the CrossMab technique (Scaefer et al. (Proc. Natl. Acad. Sci. U.S.A. (2011) 108, 11187-11192)), which is known as a technique for associating heterologous light chains that associate a light chain forming a variable region that binds to a first epitope and a light chain forming a variable region that binds to a second epitope with a heavy chain forming a variable region that binds to the first epitope and a heavy chain forming a variable region that binds to the second epitope, respectively, can also be used to produce the multispecific or multiparatopic antigen-binding molecules provided by the present invention. As a technique for producing bispecific antibodies using separately prepared monoclonal antibodies, there can be mentioned a method of promoting the heterodimerization of antibodies by placing monoclonal antibodies with specific amino acid substitutions in the heavy chain CH3 region under reducing conditions to obtain the desired bispecific antibodies. Preferred amino acid substitution sites in this method can include, for example, the residue at position 392 and the residue at position 397 in the CH3 region according to EU numbering.Furthermore, a bispecific antibody can also be prepared using an antibody in which one to three sets of amino acid residues selected from the following sets (1) to (3) in the first H-chain CH3 region have the same charge; (1) amino acid residues contained in the H-chain CH3 region, which are the amino acid residues at positions 356 and 439 in the EU numbering, (2) amino acid residues contained in the H-chain CH3 region, which are the amino acid residues at positions 357 and 370 in the EU numbering, (3) amino acid residues contained in the H-chain CH3 region, which are the amino acid residues at positions 399 and 409 in the EU numbering. Further, a set of amino acid residues selected from the sets of amino acid residues shown in (1) to (3) above in a second H-chain CH3 region different from the first H-chain CH3 region, and one to three sets of amino acid residues corresponding to the sets of amino acid residues shown in (1) to (3) above having the same charge in the first H-chain CH3 region have an opposite charge to the corresponding amino acid residues in the first H-chain CH3 region. A bispecific antibody can also be prepared using such an antibody (WO2015 / 046467).

[0080] Even when it is not possible to efficiently form the target multispecific antibody, the multispecific antibody of the present invention can also be obtained by separating and purifying the target multispecific antibody from the produced antibodies. For example, a method has been reported (WO2007114325) that enables purification of two types of homomers and the target heterologous antibody by ion exchange chromatography by introducing amino acid substitutions into the variable regions of two types of heavy chains to impart a difference in isoelectric point. Further, as a method for purifying a heteromer, a method has been reported (WO98050431, WO95033844) for purifying a heterodimeric antibody consisting of a heavy chain of mouse IgG2a that binds to Protein A and a heavy chain of rat IgG2b that does not bind to Protein A using Protein A. Furthermore, by using a heavy chain in which the 435th and 436th amino acid residues, which are the binding sites of IgG and Protein A, are substituted with amino acids having different binding affinities for Protein A, such as Tyr and His, the interaction between each heavy chain and Protein A can be changed, and only the heterodimeric antibody can be efficiently purified by using a Protein A column.

[0081] A plurality of these techniques, for example, two or more, can also be used in combination. These techniques can also be appropriately applied separately to the two heavy chains to be associated. Note that the antigen-binding molecule of the present invention may be based on the one to which the above modification has been added, and an antigen-binding molecule having the same amino acid sequence may be separately prepared.

[0082] Modification of the amino acid sequence can be carried out by various methods known in the art. These methods include, but are not limited to, site-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. Gene 152, 271-275, Zoller, MJ, and Smith, M.(1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors.Methods Enzymol. 100, 468-500, Kramer,W, Drutsa,V, Jansen,HW, Kramer,B, Pflugfelder,M, and Fritz,HJ(1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456, Kramer W, and Fritz HJ(1987) Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367, Kunkel,TA(1985) Rapid and efficient site-specific mutagenesis without phenotypic selection.Proc Natl Acad Sci U S A. 82, 488-492), PCR mutagenesis, cassette mutagenesis, and other methods.

[0083] In addition, the "antigen-binding molecule" of the present invention may be an antibody fragment that includes both the heavy chain and the light chain forming the "variable region of an antibody" of the present invention within a single polypeptide chain but lacks a constant region. Such antibody fragments may be, for example, diabody (Db), single-chain antibody, or sc(Fab')2.

[0084] Db is a dimer composed of two polypeptide chains (Holliger P et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), EP404,097, W093 / 11161, etc.). In each polypeptide chain, the L-chain variable region (VL) and the H-chain variable region (VH) are linked by a short linker, for example, about 5 residues long, such that they cannot bind to each other within the same chain. Since the linker between VL and VH encoded on the same polypeptide chain is short, they cannot form a single-chain variable region fragment. By dimerization, two antigen-binding sites are formed.

[0085] Examples of single-chain antibodies include sc(Fv)2. sc(Fv)2 is a single-chain antibody in which four variable regions, two VLs and two VHs, are linked by a linker such as a peptide linker to form a single chain (J Immunol. Methods (1999) 231 (1-2), 177-189). These two VHs and VLs may be derived from different monoclonal antibodies. For example, bispecific (bispecific sc(Fv)2) that recognizes two types of epitopes present in the same antigen as disclosed in Journal of Immunology (1994) 152 (11), 5368-5374 is also preferably mentioned. sc(Fv)2 can be produced by methods known to those skilled in the art. For example, it can be produced by linking scFv with a linker such as a peptide linker.

[0086] As the structure of the antigen-binding domains constituting sc(Fv)2 in this specification, there is an antibody characterized in that two VHs and two VLs are arranged in the order of VH, VL, VH, VL ([VH] linker [VL] linker [VH] linker [VL]) starting from the N-terminal side of a single-chain polypeptide. However, the order of the two VHs and two VLs is not particularly limited to the above structure and may be arranged in any order. For example, the following order structures can also be mentioned. [VL] linker [VH] linker [VH] linker [VL] [VH] linker [VL] linker [VL] linker [VH] [VH] linker [VH] linker [VL] linker [VL] [VL] linker [VL] linker [VH] linker [VH] [VL] linker [VH] linker [VL] linker [VH]

[0087] The molecular form of sc(Fv)2 is also described in detail in WO2006 / 132352. Those skilled in the art can appropriately prepare a desired sc(Fv)2 for the production of the antigen-binding molecules disclosed in this specification based on these descriptions.

[0088] In addition, the antigen-binding molecule of the present invention may be conjugated with a carrier polymer such as PEG or an organic compound such as an anticancer agent. Also, a sugar chain addition sequence may be inserted, and the sugar chain can be preferably added for the purpose of obtaining a desired effect.

[0089] As the linker that binds to the variable region of the antibody, any peptide linker that can be introduced by genetic engineering, or a linker disclosed in a synthetic compound linker (for example, see Protein Engineering, 9 (3), 299-305, 1996), etc. can be used, but in the present invention, a peptide linker is preferred. The length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art according to the purpose. However, the preferred length is 5 amino acids or more (the upper limit is not particularly limited, but usually 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids. When the sc(Fv)2 contains three peptide linkers, peptide linkers of all the same length may be used, or peptide linkers of different lengths may be used.

[0090] For example, in the case of a peptide linker: Ser Gly·Ser Gly·Gly·Ser Ser·Gly·Gly Gly·Gly·Gly·Ser (SEQ ID NO: 5) Ser·Gly·Gly·Gly (SEQ ID NO: 6) Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 7) Ser·Gly·Gly·Gly·Gly (SEQ ID NO: 8) Gly·Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 9) Ser·Gly·Gly·Gly·Gly·Gly (SEQ ID NO: 10) Gly·Gly·Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 11) Ser·Gly·Gly·Gly·Gly·Gly·Gly (SEQ ID NO: 12) (Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 7))n (Ser·Gly·Gly·Gly·Gly (SEQ ID NO: 8))n [n is an integer of 1 or more], etc. can be mentioned. However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art according to the purpose.

[0091] Synthetic chemical linkers (chemical crosslinking agents) are crosslinking agents commonly used for crosslinking peptides, such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl] sulfone (BSOCOES), bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl] sulfone (sulfo-BSOCOES), etc. These crosslinking agents are commercially available. When binding four antibody variable regions, usually three linkers are required, but all the same linker may be used, or different linkers may be used.

[0092] F(ab')2 contains two light chains and two heavy chains that include the constant regions of the CH1 domain and a part of the CH2 domain such that disulfide bonds between the chains are formed between the two heavy chains. The F(ab')2 constituting the polypeptide aggregate disclosed herein can be preferably obtained by partially digesting a full-length monoclonal antibody or the like having a desired antigen-binding domain with a proteolytic enzyme such as pepsin and then removing the Fc fragment by adsorption to a protein A column. Such a proteolytic enzyme is not particularly limited as long as it can digest the full-length antibody so as to restrictively produce F(ab')2 by appropriately setting the reaction conditions of the enzyme such as pH. For example, pepsin, ficin, etc. can be exemplified.

[0093] In addition to the above-mentioned amino acid modifications, the antigen-binding molecules of the present invention can further include additional modifications. The additional modifications can be selected, for example, from any of amino acid substitution, deletion, or modification, or a combination thereof. For example, modifications can be arbitrarily added to the antigen-binding molecules of the present invention as long as they do not substantially change the intended function of the molecule. For example, such mutations can be made by conservative substitution of amino acid residues. Also, even if the modification changes the intended function of the antigen-binding molecule of the present invention, such a modification can be made as long as the change in the function is within the scope of the object of the present invention.

[0094] The modification of the amino acid sequence in the present invention includes post-translational modification. As specific post-translational modifications, addition or deletion of sugar chains can be indicated. For example, when the antigen-binding molecule of the present invention has a constant region of IgG1 type, the 297th amino acid residue in EU numbering can be modified with a sugar chain. The sugar chain structure to be modified is not limited. Generally, antibodies expressed in eukaryotic cells contain sugar chain modifications in the constant region. Therefore, antibodies expressed in the following cells are usually modified with some sugar chains. · Antibody-producing cells of mammals · Eukaryotic cells transformed with an expression vector containing DNA encoding an antibody The eukaryotic cells shown here include yeast and animal cells. For example, CHO cells and HEK293H cells are typical animal cells for transformation with an expression vector containing DNA encoding an antibody. On the other hand, those without sugar chain modification at that position are also included in the antibodies of the present invention. Antibodies with a constant region not modified with a sugar chain can be obtained by expressing the gene encoding the antibody in prokaryotic cells such as Escherichia coli.

[0095] More specifically, as an additional modification in the present invention, for example, sialic acid may be added to the sugar chain in the Fc region (MAbs. 2010 Sep-Oct;2(5):519-27.).

[0096] In addition, when the antigen-binding molecule of the present invention has an Fc region portion, for example, amino acid substitutions that improve the binding activity to FcRn (J Immunol. 2006 Jan 1;176(1):346-56, J Biol Chem. 2006 Aug 18;281(33):23514-24., Int Immunol. 2006 Dec;18(12):1759-69., Nat Biotechnol. 2010 Feb;28(2):157-9., WO / 2006 / 019447, WO / 2006 / 053301, WO / 2009 / 086320), amino acid substitutions for improving antibody heterogeneity and stability ((WO / 2009 / 041613)) may be added.

[0097] Furthermore, the term "antibody" in the present invention is used in the broadest sense and includes any antibody such as monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody variants, antibody fragments, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, etc., as long as they exhibit the desired biological activity.

[0098] The antibody of the present invention is not limited by the type of antigen, the origin of the antibody, etc., and any antibody may be used. The origin of the antibody is not particularly limited, and examples include human antibodies, mouse antibodies, rat antibodies, rabbit antibodies, etc.

[0099] Methods for producing antibodies are well known to those skilled in the art. For example, in the case of monoclonal antibodies, they may be produced by the hybridoma method (Kohler and Milstein, Nature 256:495 (1975)), the recombinant method (U.S. Patent No. 4,816,567). They may also be isolated from a phage antibody library (Clackson et al., Nature 352:624-628 (1991); Marks et al., J.Mol.Biol. 222:581-597 (1991)). They may also be isolated from a single B cell clone (N. Biotechnol. 28(5): 253-457 (2011)).

[0100] Humanized antibodies are also referred to as reshaped human antibodies. Specifically, humanized antibodies such as those obtained by transplanting the CDRs of antibodies from non-human animals, for example mouse antibodies, into human antibodies are known. General genetic recombination techniques for obtaining humanized antibodies are also known. Specifically, as a method for transplanting the CDRs of mouse antibodies into human FRs, for example, Overlap Extension PCR is known.

[0101] A DNA encoding an antibody variable region in which three CDRs and four FRs are linked and a DNA encoding a human antibody constant region are inserted into an expression vector so as to be fused in-frame, whereby a vector for expressing a humanized antibody can be prepared. After introducing the integration vector into a host to establish recombinant cells, the recombinant cells are cultured and the DNA encoding the humanized antibody is expressed, whereby the humanized antibody is produced in the culture of the cultured cells (see European Patent Publication EP 239400, International Publication WO1996 / 002576).

[0102] If necessary, the amino acid residues of the FR can also be substituted so that the CDRs of the reshaped human antibody form an appropriate antigen-binding site. For example, by applying the PCR method used for transplanting mouse CDRs into human FRs, mutations in the amino acid sequence can be introduced into the FR.

[0103] Transgenic animals having all repertoires of human antibody genes (see International Publications WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, WO1996 / 033735) can be used as immunized animals, and desired human antibodies can be obtained by DNA immunization.

[0104] Furthermore, techniques for obtaining human antibodies by panning using a human antibody library are also known. For example, the V region of a human antibody is expressed on the surface of a phage by the phage display method as a single-chain antibody (scFv). Phages expressing scFv that binds to an antigen can be selected. By analyzing the gene of the selected phage, a DNA sequence encoding the V region of a human antibody that binds to the antigen can be determined. After determining the DNA sequence of scFv that binds to the antigen, an expression vector can be prepared by fusing the V region sequence in-frame with the sequence of a desired human antibody C region and then inserting it into an appropriate expression vector. The human antibody is obtained by introducing the expression vector into a suitable expression cell as described above and expressing the gene encoding the human antibody. These methods are already known (see International Publications WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, WO1995 / 015388).

[0105] In addition to the phage display method, as techniques for obtaining human antibodies by panning using a human antibody library, techniques using a cell-free translation system, techniques for presenting antigen-binding molecules on the surface of cells or viruses, techniques using emulsions, etc. are known. For example, as techniques using a cell-free translation system, there are the ribosome display method in which a complex of mRNA and a protein translated via a ribosome is formed by removing a stop codon, etc., the cDNA display method in which a gene sequence and a translated protein are covalently bonded using a compound such as puromycin, the mRNA display method, the CIS display method in which a complex of a gene and a translated protein is formed using a binding protein for a nucleic acid, etc. can be used. Further, as techniques for presenting antigen-binding molecules on the surface of cells or viruses, in addition to the phage display method, the E. coli display method, the Gram-positive bacterium display method, the yeast display method, the mammalian cell display method, the virus display method, etc. can be used. As a technique using an emulsion, the in vitro virus display method, etc. in which a gene and translation-related molecules are encapsulated in an emulsion can be used. These methods are already known (Nat Biotechnol. 2000 Dec;18(12):1287-92, Nucleic Acids Res. 2006;34(19):e127, Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):2806-10, Proc Natl Acad Sci U S A. 2004 Jun 22;101(25):9193-8, Protein Eng Des Sel. 2008 Apr;21(4):247-55, Proc Natl Acad Sci U S A. 2000 Sep 26;97(20):10701-5, MAbs. 2010 Sep-Oct;2(5):508-18, Methods Mol Biol. 2012;911:183-98).

[0106] The variable region constituting the antibody of the present invention can be a variable region that recognizes any antigen.

[0107] In this specification, "antigen" is not particularly limited and may be any antigen. Examples of antigens include 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 Adenosine Receptor, A33, ACE, ACE-2, Activin, Activin A, Activin AB, Activin B, Activin C, Activin RIA, Activin RIA ALK-2, Activin RIB ALK-4, Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, Addressins, adiponectin, ADP ribosyl cyclase-1, aFGF, AGE, ALCAM, ALK, ALK-1, ALK-7, allergen, alpha1-antichemotrypsin, alpha1-antitrypsin, alpha-synuclein, alpha-V / beta-1 antagonist, aminin, amylin, amyloid beta, amyloid immunoglobulin heavy chain variable region, amyloid immunoglobulin light chain variable region, Androgen, ANG, angiotensinogen, Angiopoietin ligand-2, anti-Id, antithrombinIII, Anthrax, APAF-1, APE, APJ, apo A1, apo serum amyloid A, Apo-SAA, APP, APRIL, AR, ARC, ART, Artemin, ASPARTIC, Atrial natriuretic factor, Atrial natriuretic peptide, atrial natriuretic peptides A, atrial natriuretic peptides Batrial natriuretic peptides C, av / b3 integrin, Axl, B7-1, B7-2, B7-H, BACE, BACE-1, Bacillus anthracis protective antigen, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, BcI, BCMA, BDNF, b-ECGF, beta-2-microglobulin, betalactamase, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, B-lymphocyte Stimulator (BIyS), BMP, BMP-2 (BMP-2a), BMP-3 (Osteogenin), BMP-4 (BMP-2b), BMP-5, BMP-6 (Vgr-1), BMP-7 (OP-1), BMP-8 (BMP-8a), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BMPR-II (BRK-3), BMPs, BOK, Bombesin, Bone-derived neurotrophic factor, bovine growth hormone, BPDE, BPDE-DNA, BRK-2, BTC, B-lymphocyte cell adhesion molecule, C10, C1-inhibitor, C1q, C3, C3a, C4, C5, C5a(complement 5a), CA125, CAD-8, Cadherin-3, Calcitonin, cAMP, Carbonic anhydrase-IX, carcinoembryonic antigen (CEA), carcinoma-associated antigen, Cardiotrophin-1, Cathepsin A, Cathepsin B, Cathepsin C / DPPI, Cathepsin D, Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O, Cathepsin S, Cathepsin V, Cathepsin X / Z / P, CBL, CCI, CCK2, CCL,CCL1 / I-309, CCL11 / Eotaxin, CCL12 / MCP-5, CCL13 / MCP-4, CCL14 / HCC-1, CCL15 / HCC-2, CCL16 / HCC-4, CCL17 / TARC, CCL18 / PARC, CCL19 / ELC, CCL2 / MCP-1, CCL20 / MIP-3-alpha, CCL21 / SLC, CCL22 / MDC, CCL23 / MPIF-1, CCL24 / Eotaxin-2, CCL25 / TECK, CCL26 / Eotaxin-3, CCL27 / CTACK, CCL28 / MEC, CCL3 / M1P-1-alpha, CCL3Ll / LD-78-beta, CCL4 / MIP-l-beta, CCL5 / RANTES, CCL6 / C10, CCL7 / MCP-3, CCL8 / MCP-2, CCL9 / 10 / MTP-1-gamma, CCR, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD10, CD105, CD11a, CD11b, CD11c, CD123, CD13, CD137, CD138, CD14, CD140a, CD146, CD147, CD148, CD15, CD152, CD16, CD164, CD18, CD19, CD2, CD20, CD21, CD22, CD23, CD25, CD26, CD27L, CD28, CD29, CD3, CD30, CD30L, CD32, CD33 (p67 proteins), CD34, CD37, CD38, CD3E, CD4, CD40, CD40L, CD44, CD45, CD46, CD49a, CD49b, CD5, CD51, CD52, CD54, CD55, CD56, CD6, CD61, CD64, CD66e, CD7, CD70, CD74, CD8, CD80 (B7-1), CD89, CD95, CD105, CD158a, CEA, CEACAM5, CFTR, cGMP, CGRP receptor, CINC, CKb8-1, Claudin18, CLC, Clostridium botulinum toxin,Clostridium difficile toxin, Clostridium perfringens toxin, c-Met, CMV, CMV UL, CNTF, CNTN-1, complement factor 3 (C3), complement factor D, corticosteroid-binding globulin, Colony stimulating factor-1 receptor, COX, C-Ret, CRG-2, CRTH2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1 / Fractalkine, CX3CR1, CXCL, CXCL1 / Gro-alpha, CXCL10, CXCL11 / I-TAC, CXCL12 / SDF-l-alpha / beta, CXCL13 / BCA-1, CXCL14 / BRAK, CXCL15 / Lungkine. CXCL16, CXCL16, CXCL2 / Gro-beta CXCL3 / Gro-gamma, CXCL3, CXCL4 / PF4, CXCL5 / ENA-78, CXCL6 / GCP-2, CXCL7 / NAP-2, CXCL8 / IL-8, CXCL9 / Mig, CXCLlO / IP-10, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cystatin C, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decay accelerating factor, Delta-like protein ligand 4, des(1-3)-IGF-1 (brain IGF-1), Dhh, DHICA oxidase, Dickkopf-1, digoxin, Dipeptidyl peptidase IV, DKl, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EGF like domain containing protein 7, Elastase, elastin, EMA, EMMPRIN, ENA,ENA-78, Endosialin, endothelin receptor, endotoxin, Enkephalinase, eNOS, Eot, Eotaxin, Eotaxin-2, eotaxini, EpCAM, Ephrin B2 / EphB4, Epha2 tyrosine kinase receptor, epidermal growth factor receptor (EGFR), ErbB2 receptor, ErbB3 tyrosine kinase receptor, ERCC, EREG, erythropoietin (EPO), Erythropoietin receptor, E-selectin, ET-1, Exodus-2, F protein of RSV, F10, F11, F12, F13, F5, F9, Factor Ia, Factor IX, Factor Xa, Factor VII, factor VIII, Factor VIIIc, Fas, FcalphaR, FcepsilonRI, FcgammaIIb, FcgammaRI, FcgammaRIIa, FcgammaRIIIa, FcgammaRIIIb, FcRn, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF-2 receptor, FGF-3, FGF-8, FGF-acidic, FGF-basic, FGFR, FGFR-3, Fibrin, fibroblast activation protein (FAP), fibroblast growth factor, fibroblast growth factor-10, fibronectin, FL, FLIP, Flt-3, FLT3 ligand, Folate receptor, follicle stimulating hormone (FSH), Fractalkine (CX3C), free heavy chain, free light chain, FZD1, FZD10, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, G250, Gas 6, GCP-2, GCSF,G-CSF, G-CSF receptor, GD2, GD3, GDF, GDF-1, GDF-15 (MIC-1), GDF-3 (Vgr-2), GDF-5 (BMP-14 / CDMP-1), GDF-6 (BMP-13 / CDMP-2), GDF-7 (BMP-12 / CDMP-3), GDF-8 (Myostatin), GDF-9, GDNF, Gelsolin, GFAP, GF-CSF, GFR-alpha1, GFR-alpha2, GFR-alpha3, GF-β1, gH envelope glycoprotein, GITR, Glucagon, Glucagon receptor, Glucagon-like peptide 1 receptor, Glut 4, Glutamate carboxypeptidase II, glycoprotein hormone receptors, glycoprotein IIb / IIIa (GP IIb / IIIa), Glypican-3, GM-CSF, GM-CSF receptor, gp130, gp140, gp72, granulocyte-CSF (G-CSF), GRO / MGSA, Growth hormone releasing factor, GRO-β, GRO-γ, H. pylori, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCC 1, HCMV gB envelope glycoprotein, HCMV UL, Hemopoietic growth factor (HGF), Hep B gp120, heparanase, heparin cofactor II, hepatic growth factor, Bacillus anthracis protective antigen, Hepatitis C virus E2 glycoprotein, Hepatitis E, Hepcidin, Her1, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HGF, HGFA,High molecular weight melanoma-associated antigen (HMW-MAA), HIV envelope proteins such as GP120, HIV MIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HMGB-1, HRG, Hrk, HSP47, Hsp90, HSV gD glycoprotein, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (hGH), human serum albumin, human tissue-type plasminogen activator (t-PA), Huntingtin, HVEM, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFN-alpha, IFN-beta, IFN-gamma, IgA, IgA receptor, IgE, IGF, IGF binding proteins, IGF-1, IGF-1 R, IGF-2, IGFBP, IGFR, IL, IL-1, IL-10, IL-10 receptors, IL-11, IL-11 receptors, IL-12, IL-12 receptors, IL-13, IL-13 receptors, IL-15, IL-15 receptors, IL-16, IL-16 receptors, IL-17, IL-17 receptors, IL-18 (IGIF), IL-18 receptors, IL-1alpha, IL-1beta, IL-1 receptors, IL-2, IL-2 receptors, IL-20, IL-20 receptors, IL-21, IL-21 receptors, IL-23, IL-23 receptors, IL-2 receptors, IL-3, IL-3 receptors, IL-31, IL-31 receptors, IL-3 receptors, IL-4, IL-4 receptors IL-5, IL-5 receptors, IL-6,IL-6 receptors, IL-7, IL-7 receptors, IL-8, IL-8 receptors, IL-9, IL-9 receptors, immunoglobulin immune complex, immunoglobulins, INF-alpha, INF-alpha receptors, INF-beta, INF-beta receptors, INF-gamma, INF-gamma receptors, IFN type-I, IFN type-I receptor, influenza, inhibin, Inhibin α, Inhibin β, iNOS, insulin, Insulin A-chain, Insulin B-chain, Insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor binding proteins, integrin, integrin alpha2, integrin alpha3, integrin alpha4, integrin alpha4 / beta1, integrin alpha-V / beta-3, integrin alpha-V / beta-6, integrin alpha4 / beta7, integrin alpha5 / beta1, integrin alpha5 / beta3, integrin alpha5 / beta6, integrin alphaσ (alphaV), integrin alphaθ, integrin beta1, integrin beta2, integrin beta3(GPIIb-IIIa), IP-10, I-TAC, JE, kalliklein, Kallikrein 11, Kallikrein 12, Kallikrein 14, Kallikrein 15, Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein L1, Kallikrein L2, Kallikrein L3, Kallikrein L4, kallistatin, KC, KDR,Keratinocyte Growth Factor (KGF), Keratinocyte Growth Factor-2 (KGF-2), KGF, killer immunoglobulin-like receptor, kit ligand (KL), Kit tyrosine kinase, laminin 5, LAMP, LAPP (Amylin, islet-amyloid polypeptide), LAP (TGF- 1), latency associated peptide, Latent TGF-1, Latent TGF-1 bp1, LBP, LDGF, LDL, LDL receptor, LECT2, Lefty, Leptin, leutinizing hormone (LH), Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, LFA-3 receptors, Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b, LTB4, LTBP-1, Lung surfactant, Luteinizing hormone, Lymphotactin, Lymphotoxin Beta Receptor, Lysosphingolipid receptor, Mac-1, macrophage-CSF (M-CSF), MAdCAM, MAG, MAP2, MARC, maspin, MCAM, MCK-2, MCP, MCP-1, MCP-2, MCP-3, MCP-4, MCP-I (MCAF), M-CSF, MDC, MDC (67 a.a.), MDC (69 a.a.), megsin, Mer, MET tyrosine kinase receptor family, METALLOPROTEASES, Membrane glycoprotein OX2, Mesothelin, MGDF receptor, MGMT, MHC (HLA-DR), microbial protein, MIF, MIG, MIP, MIP-1α, MIP-1β, MIP-3α, MIP-3β,MIP-4, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, monocyte attractant protein, monocyte colony inhibitory factor, mouse gonadotropin-associated peptide, MPIF, Mpo, MSK, MSP, MUC-16, MUC18, mucin (Mud), Muellerian-inhibiting substance, Mug, MuSK, Myelin associated glycoprotein, myeloid progenitor inhibitor factor-1 (MPIF-I), NAIP, Nanobody, NAP, NAP-2, NCA 90, NCAD, N-Cadherin, NCAM, Neprilysin, Neural cell adhesion molecule, neroserpin, Neuronal growth factor (NGF), Neurotrophin-3, Neurotrophin-4, Neurotrophin-6, Neuropilin 1, Neurturin, NGF-beta, NGFR, NKG20, N-methionyl human growth hormone, nNOS, NO, Nogo-A, Nogo receptor, non-structural protein type 3 (NS3) from the hepatitis C virus, NOS, Npn, NRG-3, NT, NT-3, NT-4, NTN, OB, OGG1, Oncostatin M, OP-2, OPG, OPN, OSM, OSM receptors, osteoinductive factors, osteopontin, OX40L, OX40R, oxidized LDL, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD,P-Cadherin, PCNA, PCSK9, PDGF, PDGF receptor, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-D, PDK-1, PECAM, PEDF, PEM, PF-4, PGE, PGF, PGI2, PGJ2, PIGF, PIN, PLA2, Placenta growth factor, placental alkaline phosphatase (PLAP), placental lactogen, pl, asminogen activator inhibitor-1, platelet-growth factor, plgR, PLP, poly glycol chains of different size(e.g. PEG-20, PEG-30, PEG40), PP14, prekallikrein, prion protein, procalcitonin, Programmed cell death protein 1, proinsulin, prolactin, Proprotein convertase PC9, prorelaxin, prostate specific membrane antigen (PSMA), Protein A, Protein C, Protein D, Protein S, Protein Z, PS, PSA, PSCA, PsmAr, PTEN, PTHrp, Ptk, PTN, P-selectin glycoprotein ligand-1, R51, RAGE, RANK, RANKL, RANTES, relaxin, Relaxin A-chain, Relaxin B-chain, renin, respiratory syncytial virus (RSV) F, Ret, reticulon 4, Rheumatoid factors, RLI P76, RPA2, RPK-1, RSK, RSV Fgp, S100, RON-8, SCF / KL, SCGF, Sclerostin, SDF-1, SDF1α, SDF1β, SERINE, Serum Amyloid P, Serum albumin, sFRP-3, Shh, Shiga like toxin II, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, sphingosine 1-phosphate receptor 1, Staphylococcal lipoteichoic acid, Stat, STEAP, STEAP-II, stem cell factor (SCF), streptokinase, superoxide dismutase,syndecan-1, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TB, TCA-3, T-cell receptor alpha / beta, TdT, TECK, TEM1, TEM5, TEM7, TEM8, Tenascin, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta Rl (ALK-5), TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, TGF-I, Thrombin, thrombopoietin (TPO), Thymic stromal lymphoprotein receptor, Thymus Ck-1, thyroid stimulating hormone (TSH), thyroxine, thyroxine-binding globulin, Tie, TIMP, TIQ, Tissue Factor, tissue factor protease inhibitor, tissue factor protein, TMEFF2, Tmpo, TMPRSS2, TNF receptor I, TNF receptor II, TNF-alpha, TNF-beta, TNF-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2 / DR4), TNFRSF10B (TRAIL R2 DR5 / KILLER / TRICK-2A / TRICK-B), TNFRSF10C (TRAIL R3 DcR1 / LIT / TRID), TNFRSF10D (TRAIL R4 DcR2 / TRUNDD), TNFRSF11A (RANK ODF R / TRANCE R), TNFRSF11B (OPG OCIF / TR1), TNFRSF12 (TWEAK R FN14), TNFRSF12A,TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR / HveA / LIGHT R / TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ / TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF Rl CD120a / p55-60), TNFRSF1B (TNF RII CD120b / p75-80), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRSF25 (DR3 Apo-3 / LARD / TR-3 / TRAMP / WSL-1), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII / TNFC R), TNFRSF4 (OX40 ACT35 / TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1 / APT1 / CD95), TNFRSF6B (DcR3 M68 / TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1 BB CD137 / ILA), TNFRST23 (DcTRAIL R1 TNFRH1), TNFSF10 (TRAIL Apo-2 Ligand / TL2), TNFSF11 (TRANCE / RANK Ligand ODF / OPG Ligand), TNFSF12 (TWEAK Apo-3 Ligand / DR3 Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS / TALL1 / THANK / TNFSF20), TNFSF14 (LIGHT HVEM Ligand / LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR Ligand AITR Ligand / TL6), TNFSF1A (TNF-a Conectin / DIF / TNFSF2), TNFSF1B (TNF-b LTa / TNFSF1), TNFSF3 (LTb TNFC / p33), TNFSF4 (OX40 Ligand gp34 / TXGP1),TNFSF5 (CD40 Ligand CD154 / gp39 / HIGM1 / IMD3 / TRAP), TNFSF6 (Fas Ligand Apo-1 Ligand / APT1 Ligand), TNFSF7 (CD27 Ligand CD70), TNFSF8 (CD30 Ligand CD153), TNFSF9 (4-1 BB Ligand CD137 Ligand), TNF-α, TNF-β, TNIL-I, toxic metabolite, TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, transforming growth factors (TGF) such as TGF-alpha and TGF-beta, Transmembrane glycoprotein NMB, Transthyretin, TRF, Trk, TROP-2, Trophoblast glycoprotein, TSG, TSLP, Tumor Necrosis Factor (TNF), tumor-associated antigen CA 125, tumor-associated antigen expressing Lewis Y related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VAP-1, vascular endothelial growth factor (VEGF), vaspin, VCAM, VCAM-1, VECAD, VE-Cadherin, VE-Cadherin-2, VEFGR-1 (flt-1), VEFGR-2, VEGF receptor (VEGFR), VEGFR-3 (flt-4), VEGI, VIM, Viral antigens, VitB12 receptor, Vitronectin receptor, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand Factor (vWF), WIF-1, WNT1, WNT10A, WNT10B, WNT11, WNT16, WNT2,Examples include WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, XCL1, XCL2 / SCM-l-beta, XCLl / Lymphotactin, XCR1, XEDAR, XIAP, XPD, etc.

[0108] Among the variable regions of the antibody contained in the antigen-binding molecule of the present invention, the "first antigen" and "second antigen" to which variable regions capable of binding to "two different antigens" but not simultaneously to these antigens bind, or the "first antigen", "second antigen" and "fourth antigen" to which variable regions capable of binding to "three different antigens" but not simultaneously to these antigens bind are, for example, antigens expressed on immune cell surface molecules (e.g., T cell surface molecules, NK cell surface molecules, dendritic cell surface molecules, B cell surface molecules, NKT cell surface molecules, MDSC cell surface molecules, macrophage surface molecules), tumor cells, tumor blood vessels, stromal cells, etc., but also expressed in normal tissues (integrin, Tissue factor, VEGFR, PDGFR, EGFR, IGFR, MET chemokine receptor, heparan sulfate proteoglycan, CD44, fibronectin, DR5, TNFRSF, etc.). As the combination of the "first antigen" and "second antigen" to which variable regions capable of binding to "two different antigens" but not simultaneously to these antigens bind, it is preferable that either one of the first antigen and the second antigen is, for example, a molecule specifically expressed on T cells, and the other antigen is a molecule expressed on the surface of T cells or other immune cells. As another aspect, as the combination of the "first antigen" and "second antigen", it is preferable that either one of the first antigen and the second antigen is, for example, a molecule specifically expressed on T cells, and the other antigen is a molecule different from the previously selected antigen expressed on immune cells. Specifically, for example, molecules specifically expressed on T cells include CD3 and T cell receptors. Particularly preferably CD3. As the site of CD3 to which the antigen-binding molecule of the present invention binds, for example, in the case of human CD3, it may bind to any epitope present in the γ-chain, δ-chain or ε-chain sequence constituting human CD3. Particularly preferred is the epitope present in the extracellular region of the ε-chain of the human CD3 complex.The structures of the γ, δ, or ε chains that make up CD3 are such that their polynucleotide sequences are described in SEQ ID NOs: 83 (NM_000073.2), 85 (NM_000732.4), and 87 (NM_000733.3), and their polypeptide sequences are described in SEQ ID NOs: 84 (NP_000064.1), 86 (NP_000723.1), and 88 (NP_000724.1) (the numbers in parentheses indicate RefSeq accession numbers). Further examples of other antigens include Fcγ receptors, TLRs, lectins, IgA, immune checkpoint molecules, TNF superfamily molecules, TNFR superfamily molecules, and NK receptor molecules. In addition, as a combination of the "first antigen", "second antigen", and "fourth antigen" to which the variable region that can bind to "three different antigens" but cannot bind to these antigens simultaneously binds, it is preferable that any one of the first antigen, the second antigen, and the fourth antigen is, for example, a molecule specifically expressed on T cells, and the remaining two antigens are molecules expressed on the surface of T cells or other immune cells. As another aspect, as a combination of the "first antigen", "second antigen", and "fourth antigen", it is preferable that any one of the first antigen, the second antigen, and the fourth antigen is, for example, a molecule specifically expressed on T cells, and the remaining two antigens are molecules expressed on immune cells and different from the previously selected antigen. Specifically, for example, molecules specifically expressed on T cells include CD3 and T cell receptors. Particularly preferably CD3. As the site of CD3 to which the antigen-binding molecule of the present invention binds, for example, in the case of human CD3, it may bind to any epitope as long as it is an epitope present in the γ-chain, δ-chain, or ε-chain sequence constituting human CD3. Particularly preferred is an epitope present in the extracellular region of the ε-chain of the human CD3 complex. The structures of the γ-chain, δ-chain, or ε-chain constituting CD3 are such that their polynucleotide sequences are described in SEQ ID NOs: 83 (NM_000073.2), 85 (NM_000732.4), and 87 (NM_000733.3), and their polypeptide sequences are described in SEQ ID NOs: 84 (NP_000064.1), 86 (NP_000723.1), and 88 (NP_000724.1) (the numbers in parentheses indicate RefSeq accession numbers). Further, the remaining two antigens include Fcγ receptor, TLR, lectin, IgA, immune checkpoint molecule, TNF superfamily molecule, TNFR superfamily molecule, and NK receptor molecule. In addition, as the "third antigen" to which the other variable region among the variable regions of the antibody contained in the antigen-binding molecule of the present invention binds, which is different from the above-mentioned "first antigen", "second antigen", and "fourth antigen", for example, an antigen specific to tumor cells is preferable. In addition to the antigen that is expressed as the cell becomes malignant, it also includes abnormal sugar chains that appear on the cell surface or protein molecules when the cell becomes cancerous. Specifically, for example, ALK receptor (pleiotrophin receptor), pleiotrophin, KS 1 / 4 pancreatic cancer antigen, ovarian cancer antigen (CA125), prostate acid phosphate, prostate-specific antigen (PSA), melanoma-related antigen p97, melanoma antigen gp75, high molecular weight melanoma antigen (HMW-MAA), prostate-specific membrane antigen, carcinoembryonic antigen (CEA), polymorphic epithelial mucin antigen, human milk fat globule antigen, CEA, TAG-72, CO17-1A, GICA 19-9, colorectal tumor-related antigens such as CTA-1 and LEA, Burkitt lymphoma antigen-38.13, melanoma-specific antigens such as CD19, human B-lymphoma antigen-CD20, CD33, ganglioside GD2, ganglioside GD3, ganglioside GM2 and ganglioside GM3, tumor-specific transplantation-type cell surface antigens (TSTA), T antigen, tumor antigens induced by viruses such as envelope antigens of DNA tumor viruses and RNA tumor viruses, carcinoembryonic antigens such as CEA of the colon, 5T4 carcinoembryonic trophoblast glycoprotein and carcinoembryonic antigen of bladder tumors, α-fetoprotein, differentiation antigens such as human lung cancer antigens L6 and L20, antigens of fibrosarcoma, human leukemia T cell antigen-Gp37, neonatal glycoproteins, sphingolipids, breast cancer antigens such as EGFR (epidermal growth factor receptor), NY-BR-16, NY-BR-16 and HER2 antigen (p185HER2), polymorphic epithelial mucin (PEM), malignant human lymphocyte antigen-APO-1, differentiation antigens such as I antigen found on fetal erythrocytes, early endodermal I antigen found on adult erythrocytes, I(Ma) found in gastric cancer, M18, M39 found in mammary epithelium, SSEA-1 found in bone marrow cells, VEP8, VEP9, Myl, VIM-D5, D156-22 found in colorectal cancer, TRA-1-85 (blood group H), SCP-1 found in testicular and ovarian cancers, C14 found in colon cancer, F3 found in lung cancer, AH6 found in gastric cancer, Y hapten, Ley found in embryonal carcinoma cells, TL5 (blood group A), EGF receptor found in A431 cells, E1 series (blood group B) found in pancreatic cancer, FC10.2 found in embryonal carcinoma cells, gastric cancer antigen, CO-514 (blood group Lea) found in adenocarcinoma, NS-10 found in adenocarcinoma, CO-43 (blood group Leb), G49 found on the EGF receptor of A431 cells, MH2 (blood group ALeb / Ley) found in colon cancer, 19.9 found in colon cancer, gastric cancer mucin, T5A7 found in bone marrow cells, R24 found in melanoma, 4.2 found in embryonal carcinoma cells, GD3, D1.1. OFA-1, GM2, OFA-2, GD2, and M1:22:25:8, as well as SSEA-3 and SSEA-4 found in 4- to 8-cell stage embryos, subcutaneous T cell lymphoma antigen, MART-1 antigen, sialyl Tn (STn) antigen, colon cancer antigen NY-CO-45, lung cancer antigen NY-LU-12 variant A, adenocarcinoma antigen ART1, tumor-associated testicular cancer antigen (cancer neural antigen MA2, tumor-associated neural antigen), neural cancer abdominal antigen 2 (NOVA2), blood cell cancer antigen gene 520, tumor-associated antigen CO-029, tumor-associated antigen MAGE-C1 (cancer / testis antigen CT7), MAGE-B1 (MAGE-XP antigen), MAGE-B2 (DAM6), MAGE-2, MAGE-4a, MAGE-4b, and MAGE-X2, cancer / testis antigen (NY-EOS-1), YKL-40, and any fragment of the above polypeptides or a structure modified thereto (such as the modified phosphate groups and sugar chains described above), EpCAM, EREG, CA19-9, CA15-3, serial SSEA-1 (SLX), HER2, PSMA, CEA, CLEC12A, etc.

[0109] The antigen-binding molecules of the present invention can be produced by methods known to those skilled in the art. For example, antibodies can be produced by the following methods, but are not limited thereto. Many combinations of host cells and expression vectors for producing antibodies by introducing a gene encoding an isolated polypeptide into an appropriate host are known. Any of these expression systems can be applied to isolate the antigen-binding molecules of the present invention. When eukaryotic cells are used as host cells, animal cells, plant cells, or fungal cells can be appropriately used. Specifically, examples of animal cells can include the following cells. (1) Mammalian cells: CHO (Chinese hamster ovary cell line), COS (Monkey kidney cell line), myeloma (Sp2 / O, NS0, etc.), BHK (baby hamster kidney cell line), HEK293 (human embryonic kidney cell line with sheared adenovirus (Ad)5 DNA), PER.C6 cell (human embryonic retinal cell line transformed with the Adenovirus Type 5 (Ad5) E1A and E1B genes), Hela, Vero, etc. (Current Protocols in Protein Science (May, 2001, Unit 5.9, Table 5.9.1)) (2) Amphibian cells: African clawed frog oocytes, etc. (3) Insect cells: sf9, sf21, Tn5, etc. Also, antibodies can be produced in Escherichia coli (mAbs 2012 Mar - Apr; 4(2): 217 - 225.) or yeast (WO2000023579). Antibodies produced in Escherichia coli do not have sugar chains added. On the other hand, antibodies produced in yeast have sugar chains added.

[0110] DNA encoding the heavy chain of an antibody, which encodes a heavy chain in which one or more amino acid residues in the variable region are substituted with other amino acids of interest, and DNA encoding the light chain of the antibody are expressed. DNA encoding a heavy chain or light chain in which one or more amino acid residues in the variable region are substituted with other amino acids of interest can be obtained, for example, by obtaining DNA encoding the variable region of an antibody produced using a known method against a certain antigen, and appropriately introducing substitutions so that the codon encoding a specific amino acid in the region encodes the other amino acid of interest.

[0111] Alternatively, DNA encoding a protein in which one or more amino acid residues in the variable region of an antibody prepared in advance using a known method against a certain antigen are substituted with other target amino acids is designed, and by chemically synthesizing the DNA, it is also possible to obtain DNA encoding a heavy chain in which one or more amino acid residues in the variable region are substituted with other target amino acids. The substitution site of the amino acid and the type of substitution are not particularly limited. Preferred regions for amino acid modification include regions exposed to the solvent and loop regions in the variable region. Among them, CDR1, CDR2, CDR3, FR3 regions, and loop regions are preferred. Specifically, Kabat numbering 31 - 35, 50 - 65, 71 - 74, 95 - 102 of the H-chain variable region and Kabat numbering 24 - 34, 50 - 56, 89 - 97 of the L-chain variable region are preferred, and Kabat numbering 31, 52a - 61, 71 - 74, 97 - 101 of the H-chain variable region and Kabat numbering 24 - 34, 51 - 56, 89 - 96 of the L-chain variable region are more preferred. Also, the amino acid modification is not limited to substitution, and may be any of deletion, addition, insertion, or modification, or a combination thereof.

[0112] In addition, DNA encoding a heavy chain in which one or more amino acid residues in the variable region are substituted with other target amino acids can be produced in divided partial DNAs. Examples of combinations of partial DNAs include, but are not limited to, DNA encoding the variable region and DNA encoding the constant region, or DNA encoding the Fab region and DNA encoding the Fc region. DNA encoding the light chain can also be produced in divided partial DNAs in the same manner.

[0113] Examples of methods for expressing the above DNA include the following. For example, DNA encoding a heavy chain variable region is incorporated into an expression vector together with DNA encoding a heavy chain constant region to construct a heavy chain expression vector. Similarly, DNA encoding a light chain variable region is incorporated into an expression vector together with DNA encoding a light chain constant region to construct a light chain expression vector. These heavy chain and light chain genes can also be incorporated into a single vector.

[0114] When incorporating the DNA encoding the target antibody into an expression vector, it is incorporated into the expression vector so as to be expressed under the control of an expression control region, for example, an enhancer or a promoter. Next, the host cell is transformed with this expression vector to express the antibody. In this case, an appropriate combination of host and expression vector can be used.

[0115] Examples of vectors include M13-based vectors, pUC-based vectors, pBR322, pBluescript, pCR-Script, etc. Also, when the purpose is cDNA subcloning or excision, in addition to the above vectors, for example, pGEM-T, pDIRECT, pT7, etc. can be used.

[0116] When using a vector for the purpose of producing the antibody of the present invention, in particular, an expression vector is useful. As the expression vector, for example, when the host is Escherichia coli such as JM109, DH5α, HB101, XL1-Blue, etc., a promoter capable of efficient expression in Escherichia coli, for example, the lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427, the entire content of which is incorporated herein by reference), the araB promoter (Better et al., Science (1988) 240, 1041-1043, the entire content of which is incorporated herein by reference), or the T7 promoter, etc. are essential. Such vectors include, in addition to the above vectors, pGEX-5X-1 (manufactured by Pharmacia), "QIAexpress system" (manufactured by QIAGEN), pEGFP, or pET (in this case, the host is preferably BL21 expressing T7 RNA polymerase), etc.

[0117] In addition, the vector may contain a signal sequence for polypeptide secretion. As the signal sequence for polypeptide secretion, when producing in the periplasm of Escherichia coli, the pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4397, the entire content of which is incorporated herein by reference) may be used. Introduction of the vector into the host cell can be carried out using, for example, the lipofectin method, the calcium phosphate method, or the DEAE-Dextran method.

[0118] In addition to the Escherichia coli expression vector, for example, as vectors for producing the polypeptide of the present invention, expression vectors derived from mammals (for example, pcDNA3 (manufactured by Invitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18(17), p5322, the entire content of which is incorporated herein by reference), pEF, pCDM8), expression vectors derived from insect cells (for example, "Bac-to-BAC baculovirus expression system" (manufactured by GIBCO BRL), pBacPAK8), expression vectors derived from plants (for example, pMH1, pMH2), expression vectors derived from animal viruses (for example, pHSV, pMV, pAdexLcw), expression vectors derived from retroviruses (for example, pZIPneo), expression vectors derived from yeast (for example, "Pichia Expression Kit" (manufactured by Invitrogen), pNV11, SP-Q01), and expression vectors derived from Bacillus subtilis (for example, pPL608, pKTH50) can be mentioned.

[0119] When aiming for expression in animal cells such as CHO cells, COS cells, NIH3T3 cells, HEK293 cells, etc., it is essential to have promoters necessary for intracellular expression, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108, which is hereby incorporated by reference in its entirety), MMTV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322, which is hereby incorporated by reference in its entirety), CAG promoter (Gene. (1991) 108, 193, which is hereby incorporated by reference in its entirety), CMV promoter, etc. It is more preferable to have a gene for selecting transformed cells (for example, a drug resistance gene that can be discriminated by a drug (such as neomycin, G418)). Examples of vectors having such characteristics include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOP13, etc. In some cases, EBNA1 protein may be co-expressed for the purpose of increasing the gene copy number. In this case, a vector having the replication origin OriP is used. (Biotechnol Bioeng. 2001 Oct 20;75(2):197-203., Biotechnol Bioeng. 2005 Sep 20;91(6):670-7.)

[0120] Furthermore, when the aim is to stably express a gene and amplify the copy number of the gene in cells, a method can be used in which a vector having a DHFR gene (e.g., pCHOI, etc.) that complements CHO cells lacking the nucleic acid synthesis pathway is introduced and amplified with methotrexate (MTX). When the aim is transient expression of a gene, a method can be used in which COS cells having a gene expressing SV40 T antigen on the chromosome are transformed with a vector (e.g., pcD, etc.) having an SV40 origin of replication. As the origin of replication, those derived from polyomavirus, adenovirus, bovine papillomavirus (BPV), etc. can also be used. Furthermore, for gene copy number amplification in the host cell line, the expression vector can contain, as a selectable marker, an aminoglycoside transferase (APH) gene, a thymidine kinase (TK) gene, an Escherichia coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, a dihydrofolate reductase (dhfr) gene, etc.

[0121] Recovery of the antibody can be carried out, for example, by culturing the transformed cells and then separating them from inside the cells or the culture solution of the molecularly transformed cells. For separation and purification of the antibody, methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, C1q, FcRn, protein A, protein G columns, affinity chromatography, ion exchange chromatography, gel filtration chromatography, etc. can be appropriately combined and carried out.

[0122] As an efficient method for producing a multispecific antibody, the aforementioned techniques such as the Knobs-into-holes technique (WO1996 / 027011, Ridgway JB et al., Protein Engineering (1996) 9, 617-621, Merchant AM et al. Nature Biotechnology (1998) 16, 677-681) and the technique of introducing a charge repulsion to suppress the association of non-target H chains (WO2006 / 106905) can be applied.

[0123] Furthermore, the present invention provides a method for producing an antigen-binding molecule, which comprises a variable region of an antibody capable of binding to two different first antigens and a second antigen, the variable region not binding to the first antigen and the second antigen simultaneously (first variable region), and a variable region capable of binding to a third antigen different from the first antigen and the second antigen (second variable region), and includes a step of creating a library of antigen-binding molecules with diverse amino acid sequences of the first variable region.

[0124] For example, a production method including the following steps can be mentioned: (i) A step of creating a library of antigen-binding molecules in which at least one amino acid of the variable region of an antibody that binds to the first antigen or the second antigen is modified, and the variable region contains at least one amino acid that is different from each other. (ii) A step of selecting, from the created library, an antigen-binding molecule that has binding activity to the first antigen and the second antigen but does not bind to the first antigen and the second antigen simultaneously. (iii) Culturing a host cell containing a nucleic acid encoding the variable region of the antigen-binding molecule selected in step (ii) and a nucleic acid encoding the variable region of an antigen-binding molecule that binds to the third antigen, to express an antigen-binding molecule that can bind to the first antigen and the second antigen but does not bind to the first antigen and the second antigen simultaneously, and a variable region that binds to the third antigen, and (iv) A step of recovering the antigen-binding molecule from the host cell culture.

[0125] In this production method, step (ii) may be the following selection step: (v) A step of selecting, from the created library, an antigen-binding molecule that has binding activity to the first antigen and the second antigen but does not bind to the first antigen and the second antigen expressed on different cells simultaneously.

[0126] The antigen-binding molecule used in the above step (i) is not particularly limited as long as it contains the variable region of an antibody, and may be an antibody fragment such as Fv, Fab, Fab', or an antibody containing an Fc region.

[0127] As the amino acid to be modified, for example, among the variable regions of the antibody that binds to the first antigen or the second antigen, an amino acid that does not lose its binding to the antigen due to amino acid modification is selected.

[0128] The amino acid modification of the present invention may be used alone or in combination of a plurality. When used in combination of a plurality, the number of combinations is not particularly limited. For example, it is 2 or more and 30 or less, preferably 2 or more and 25 or less, 2 or more and 22 or less, 2 or more and 20 or less, 2 or more and 15 or less, 2 or more and 10 or less, 2 or more and 5 or less, 2 or more and 3 or less. When combining a plurality, the amino acid modification may be added only to the variable region of the heavy chain or the light chain of the antibody, or may be appropriately distributed and added to both the variable region of the heavy chain and the variable region of the light chain.

[0129] Preferred regions for amino acid modification include regions exposed to the solvent and loop regions in the variable region. Among them, CDR1, CDR2, CDR3, FR3 regions, and loop regions are preferred. Specifically, Kabat numbering 31-35, 50-65, 71-74, 95-102 of the heavy chain variable region and Kabat numbering 24-34, 50-56, 89-97 of the light chain variable region are preferred, and Kabat numbering 31, 52a-61, 71-74, 97-101 of the heavy chain variable region and Kabat numbering 24-34, 51-56, 89-96 of the light chain variable region are more preferred.

[0130] In addition, modifying the amino acid residues includes randomly modifying the amino acids in the above-described region of the variable region of the antibody that binds to the first antigen or the second antigen, or inserting a peptide that is known to have binding activity to a desired antigen in advance into the above-described region. In this way, among the antigen-binding molecules with modifications, by selecting a variable region that can bind to the first antigen and the second antigen but cannot bind to these antigens simultaneously, it is possible to obtain the antigen-binding molecule of the present invention. Examples of peptides that are known to have binding activity to a desired antigen in advance include the peptides shown in Table 1 above.

[0131] Whether it is a variable region that can bind to the first antigen and the second antigen but cannot bind to these antigens simultaneously, and further, when either the first antigen or the second antigen is present on the cell surface and the other is present alone, both are present alone, or both are present on the same cell surface, whether it is a variable region that can bind to both the first antigen and the second antigen simultaneously, but when expressed on different cells, cannot bind simultaneously can be confirmed in the same manner according to the above-described method.

[0132] Furthermore, the present invention provides a method for producing an antigen-binding molecule, which comprises a variable region of an antibody that can bind to two different first antigens and second antigens and does not bind to the first antigen and the second antigen simultaneously (the first variable region), and includes a step of preparing a library of antigen-binding molecules with diverse amino acid sequences of the first variable region.

[0133] As a method for producing such an antigen-binding molecule, for example, a production method including the following steps can be mentioned: (i) A step of preparing a library of antigen-binding molecules, which are antigen-binding molecules in which at least one amino acid of the variable region of an antibody that binds to the first antigen or the second antigen is modified, and the at least one amino acid of the modified variable region is different from each other. (ii) Selecting, from the prepared library, an antigen-binding molecule that has binding activity against the first antigen and the second antigen but does not bind to the first antigen and the second antigen simultaneously and that contains a variable region; (iii) Culturing a host cell containing a nucleic acid encoding the variable region of the antigen-binding molecule selected in step (ii) to express an antigen-binding molecule that can bind to the first antigen and the second antigen but does not bind to the first antigen and the second antigen simultaneously and that contains a variable region of an antibody; and (iv) Recovering the antigen-binding molecule from the host cell culture. Note that, as a preferable region for the above amino acid modification, the heavy-chain variable region can be mentioned. More preferably, regions exposed to the solvent and loop regions in the variable region can be mentioned. Among them, CDR1, CDR2, CDR3, FR3 regions, and loop regions are preferable. Specifically, Kabat numbering 31-35, 50-65, 71-74, 95-102 of the H-chain variable region and Kabat numbering 24-34, 50-56, 89-97 of the L-chain variable region are preferable, and Kabat numbering 31, 52a-61, 71-74, 97-101 of the H-chain variable region and Kabat numbering 24-34, 51-56, 89-96 of the L-chain variable region are more preferable.

[0134] Note that, in this production method, step (ii) may be the following selection step: (v) Selecting, from the prepared library, an antigen-binding molecule that has binding activity against the first antigen and the second antigen but does not bind to the first antigen and the second antigen expressed on different cells simultaneously and that contains a variable region;

[0135] The antigen-binding molecule used in step (i) above is not particularly limited as long as it contains the variable region of an antibody, and may be an antibody fragment such as Fv, Fab, Fab', or an antibody containing an Fc region.

[0136] As the amino acids to be modified, for example, among the variable regions of the antibody that binds to the first antigen or the second antigen, amino acids that do not lose the binding to the antigen due to amino acid modification are selected.

[0137] The amino acid modifications of the present invention may be used alone or in combination of multiple ones. When used in combination of multiple ones, the number of combinations is not particularly limited. For example, it is 2 or more and 30 or less, preferably 2 or more and 25 or less, 2 or more and 22 or less, 2 or more and 20 or less, 2 or more and 15 or less, 2 or more and 10 or less, 2 or more and 5 or less, 2 or more and 3 or less. When combining multiple ones, the amino acid modification may be added to only one of the heavy chain variable region or the light chain variable region of the antibody, or may be appropriately distributed and added to both the heavy chain variable region and the light chain variable region.

[0138] In addition, adding a modification to an amino acid residue includes randomly modifying the amino acids in the above-mentioned region among the variable regions of the antibody that binds to the first antigen or the second antigen, or inserting a peptide known to have binding activity to a desired antigen into the above-mentioned region. In this way, among the antigen-binding molecules with modifications, by selecting a variable region that can bind to the first antigen and the second antigen but cannot bind to these antigens simultaneously, the antigen-binding molecule of the present invention can be obtained. Examples of peptides known to have binding activity to a desired antigen include the peptides shown in Table 1 above.

[0139] Whether it is a variable region that can bind to the first antigen and the second antigen but cannot bind to these antigens simultaneously, and further, when one of the first antigen and the second antigen exists on the cell surface and the other exists alone, when both exist alone, or when both exist on the same cell, it can bind to both the first antigen and the second antigen simultaneously, but when they are expressed on different cells, whether it is a variable region that cannot bind simultaneously can be confirmed in the same way according to the above method.

[0140] Furthermore, the antigen-binding molecules produced by the production method are also included in the present invention. The types and ranges of amino acid modifications introduced by this method are not particularly limited.

[0141] As a non-limiting aspect of the library of the present invention, CD3 (in the case of human CD3, the γ chain, δ chain, or ε chain constituting human CD3) is selected as the first antigen, and a library consisting of antigen-binding molecules that bind to CD3 and any second antigen can be mentioned.

[0142] As a preferred aspect of the library of the present invention, a library mainly composed of a plurality of antigen-binding molecules having different sequences, wherein the antigen-binding regions in the antigen-binding molecules can bind to a first antigen and a second antigen different from the first antigen, but are variable regions of an antibody that do not bind to the first antigen and the second antigen simultaneously, and either one of the first antigen and the second antigen is CD3, and the other antigen is a molecule expressed on the surface of T cells or other immune cells.

[0143] When either one of the first antigen and the second antigen is human CD3, the antigen-binding molecule preferably binds to the γ chain, δ chain, or ε chain constituting human CD3. Furthermore, it is preferable that the variable region is a variable region that does not bind to the first antigen and the second antigen expressed on different cells simultaneously. Here, "expressed on different cells" means that it may be expressed on separate cells, and examples of such cell combinations may be the same type of cells such as T cells and another T cell, or different types of cells such as T cells and NK cells.

[0144] Furthermore, the present invention provides a method for producing an antigen-binding molecule, which comprises a variable region of an antibody capable of binding to three different antigens (a first antigen, a second antigen different from the first antigen, and a fourth antigen different from the first antigen and the second antigen), the variable region not binding to the three antigens simultaneously (a first variable region), and a variable region binding to a third antigen different from the three antigens (a second variable region), and the method includes a step of creating a library of antigen-binding molecules with diverse amino acid sequences of the first variable region.

[0145] For example, a production method including the following steps can be mentioned: (i) Creating a library of antigen-binding molecules, which is an antigen-binding molecule in which at least one amino acid of the variable region of an antibody binding to three different antigens (a first antigen, a second antigen different from the first antigen, and a fourth antigen different from the first antigen and the second antigen) is modified, and the variable region contains at least one amino acid of the modified variable region that is different from each other. (ii) Selecting, from the created library, an antigen-binding molecule containing a variable region that has binding activity to the three antigens but does not bind to the three antigens simultaneously. (iii) Culturing a host cell containing a nucleic acid encoding the variable region of the antigen-binding molecule selected in step (ii) and a nucleic acid encoding the variable region of an antigen-binding molecule that binds to a third antigen, to express an antigen-binding molecule containing a variable region that can bind to the three antigens but does not bind to the three antigens simultaneously, and a variable region that binds to the third antigen, and (iv) Recovering the antigen-binding molecule from the host cell culture.

[0146] In this production method, step (ii) may be the following selection step: (v) Selecting, from the created library, an antigen-binding molecule containing a variable region that has binding activity to the three antigens but does not bind to the three antigens expressed on different cells simultaneously.

[0147] The antigen-binding molecule used in the above step (i) is not particularly limited as long as it contains the variable region of an antibody, and may be an antibody fragment such as Fv, Fab, Fab', or an antibody containing an Fc region.

[0148] As the amino acid to be modified, for example, among the variable regions of the antibodies that bind to the three antigens, an amino acid that does not lose its binding to the antigen due to amino acid modification is selected.

[0149] The amino acid modifications of the present invention may be used alone or in combination of multiple ones. When used in combination of multiple ones, the number of combinations is not particularly limited. For example, it is 2 or more and 30 or less, preferably 2 or more and 25 or less, 2 or more and 22 or less, 2 or more and 20 or less, 2 or more and 15 or less, 2 or more and 10 or less, 2 or more and 5 or less, 2 or more and 3 or less. When combining multiple ones, the amino acid modification may be added only to the variable region of the heavy chain or the light chain of the antibody, or may be appropriately distributed and added to both the variable region of the heavy chain and the variable region of the light chain.

[0150] Preferred regions for amino acid modification include regions exposed to the solvent and loop regions in the variable region. Among them, CDR1, CDR2, CDR3, FR3 regions, and loop regions are preferred. Specifically, Kabat numbering 31 - 35, 50 - 65, 71 - 74, 95 - 102 of the H-chain variable region and Kabat numbering 24 - 34, 50 - 56, 89 - 97 of the L-chain variable region are preferred, and Kabat numbering 31, 52a - 61, 71 - 74, 97 - 101 of the H-chain variable region and Kabat numbering 24 - 34, 51 - 56, 89 - 96 of the L-chain variable region are more preferred.

[0151] In addition, modifying the amino acid residues includes randomly modifying the amino acids in the above-described region among the variable regions of the antibodies that bind to the three antigens, or inserting a peptide known in advance to have binding activity to a desired antigen into the above-described region. In this way, from among the antigen-binding molecules to which modifications have been made, by selecting a variable region that can bind to three different antigens (a first antigen, a second antigen different from the first antigen, and a fourth antigen different from the first antigen and the second antigen) but cannot bind to these antigens simultaneously, it is possible to obtain the antigen-binding molecule of the present invention. Examples of peptides known in advance to have binding activity to a desired antigen include the peptides shown in Table 1 above.

[0152] Whether it is a variable region that can bind to the three antigens but cannot bind to these antigens simultaneously, and further, when any one or more of the three different antigens (first antigen, second antigen, fourth antigen) exist alone rather than on the cell surface, or when any two or more of the three different antigens are present on the same cell, they can bind simultaneously with respect to those antigens, but when expressed on different cells, whether it is a variable region that cannot bind simultaneously can be confirmed in the same manner according to the above-described method.

[0153] Furthermore, the present invention provides a method for producing an antigen-binding molecule, which is a variable region of an antibody that can bind to three different antigens (a first antigen, a second antigen different from the first antigen, and a fourth antigen different from the first antigen and the second antigen), and includes a variable region (first variable region) that does not bind to the three antigens simultaneously, and includes a step of creating a library of diverse antigen-binding molecules with the amino acid sequence of the first variable region.

[0154] As a method for producing such an antigen-binding molecule, for example, a production method including the following steps can be mentioned: (i) A step of preparing a library of antigen-binding molecules in which at least one amino acid of the variable region of an antibody that binds to three different antigens (a first antigen, a second antigen different from the first antigen, and a fourth antigen different from the first antigen and the second antigen) is modified, and at least one of the amino acids of the modified variable region contains variable regions that are different from each other. (ii) A step of selecting, from the prepared library, an antigen-binding molecule containing a variable region that has binding activity to the three antigens but does not bind to the three antigens simultaneously. (iii) A step of culturing a host cell containing a nucleic acid encoding the variable region of the antigen-binding molecule selected in step (ii) to express an antigen-binding molecule containing a variable region of an antibody that can bind to the three antigens but does not bind to the three antigens simultaneously, and (iv) A step of recovering the antigen-binding molecule from the host cell culture. Note that preferred regions for the above amino acid modification include the heavy chain variable region. More preferably, regions exposed to the solvent and loop regions in the variable region are included. Among them, CDR1, CDR2, CDR3, FR3 regions, and loop regions are preferred. Specifically, Kabat numbering 31 - 35, 50 - 65, 71 - 74, 95 - 102 of the H chain variable region and Kabat numbering 24 - 34, 50 - 56, 89 - 97 of the L chain variable region are preferred, and Kabat numbering 31, 52a - 61, 71 - 74, 97 - 101 of the H chain variable region and Kabat numbering 24 - 34, 51 - 56, 89 - 96 of the L chain variable region are more preferred.

[0155] Note that in this production method, step (ii) above may be the following selection step: (v) A step of selecting, from the prepared library, an antigen-binding molecule containing a variable region that has binding activity to the three antigens but does not bind to the three antigens expressed on different cells simultaneously.

[0156] The antigen-binding molecule used in the above step (i) is not particularly limited as long as it contains the variable region of an antibody, and may be an antibody fragment such as Fv, Fab, Fab', or an antibody containing an Fc region.

[0157] As the amino acid to be modified, for example, among the variable regions of the antibody that binds to the three antigens, an amino acid that does not lose its binding to the antigen due to amino acid modification is selected.

[0158] The amino acid modifications of the present invention may be used alone or in combination of multiple ones. When used in combination of multiple ones, the number of combinations is not particularly limited. For example, it is 2 or more and 30 or less, preferably 2 or more and 25 or less, 2 or more and 22 or less, 2 or more and 20 or less, 2 or more and 15 or less, 2 or more and 10 or less, 2 or more and 5 or less, 2 or more and 3 or less. When combining multiple ones, the amino acid modification may be added to only one of the heavy chain variable region or the light chain variable region of the antibody, or may be appropriately distributed and added to both the heavy chain variable region and the light chain variable region.

[0159] In addition, adding a modification to an amino acid residue includes randomly modifying the amino acids in the above-mentioned regions among the variable regions of the antibody that binds to the three antigens, or inserting a peptide known to have binding activity to a desired antigen into the above-mentioned regions. In this way, among the antigen-binding molecules with modifications added, a variable region that can bind to the three antigens but cannot bind to these antigens simultaneously is selected, and thus it is possible to obtain the antigen-binding molecule of the present invention. Examples of peptides known to have binding activity to a desired antigen in advance include the peptides shown in Table 1 above.

[0160] Whether it has variable regions that can bind to the three antigens but cannot bind to these antigens simultaneously, and further, when any one or more of the three different antigens (the first antigen, the second antigen, and the fourth antigen) exist alone rather than on cells, or when any two or more of the three different antigens exist on the same cell, they can bind simultaneously with respect to those antigens, but when they are expressed on different cells, whether they have variable regions that cannot bind simultaneously can be similarly confirmed according to the above-described method.

[0161] Furthermore, antigen-binding molecules produced by the production method are also included in the present invention. The types and ranges of amino acid modifications introduced by this method are not particularly limited.

[0162] As a non-limiting aspect of the library of the present invention, there can be mentioned a library consisting of antigen-binding molecules that select CD3 (in the case of human CD3, the γ chain, δ chain, or ε chain constituting human CD3) as the first antigen and bind to CD3 and any second antigen and fourth antigen.

[0163] As a preferred aspect of the library of the present invention, there can be mentioned a library mainly consisting of a plurality of antigen-binding molecules having different sequences, wherein the antigen-binding regions in the antigen-binding molecules are variable regions of an antibody that can bind to three different antigens (the first antigen, a second antigen different from the first antigen, and a fourth antigen different from the first antigen and the second antigen) but do not bind to the three antigens simultaneously, and any one of the third antigens is CD3, and the remaining two antigens are molecules expressed on the surface of T cells or other immune cells.

[0164] When any one of the three antigens is human CD3, the antigen-binding molecule preferably binds to the γ chain, δ chain, or ε chain that constitutes human CD3. Further, it is preferable that the variable regions are variable regions that do not simultaneously bind to the three antigens expressed on different cells. Here, "expressed on different cells" means that it may be expressed on separate cells, and examples of such combinations of cells may be the same type of cells, such as T cells and another T cell, or different types of cells, such as T cells and NK cells.

[0165] As used herein, "library" or "library" refers to a plurality of antigen-binding molecules or a plurality of fusion polypeptides containing antigen-binding molecules, or nucleic acids or polynucleotides encoding these sequences. The sequences of the plurality of antigen-binding molecules or the plurality of fusion polypeptides containing antigen-binding molecules contained in the library are not a single sequence, but antigen-binding molecules with different sequences from each other or fusion polypeptides containing antigen-binding molecules.

[0166] In one embodiment of the present invention, a fusion polypeptide of the antigen-binding molecule of the present invention and a heterologous polypeptide can be prepared. In certain embodiments, the fusion polypeptide can be fused with at least a portion of a viral coat protein selected from the group consisting of viral coat proteins such as pIII, pVIII, pVII, pIX, Soc, Hoc, gpD, pVI, and variants thereof.

[0167] In certain embodiments, the antigen-binding molecule of the present invention can be a ScFv, Fab fragment, F(ab)2 or F(ab')2. Thus, in another embodiment, a library consisting mainly of a plurality of fusion polypeptides having different sequences of these antigen-binding molecules and a heterologous polypeptide is provided. Specifically, a library consisting mainly of a plurality of fusion polypeptides having different sequences, which are fusion polypeptides of these antigen-binding molecules and at least a part of a viral coat protein selected from the group consisting of viral coat proteins such as pIII, pVIII, pVII, pIX, Soc, Hoc, gpD, pVI and variants thereof, is provided. The antigen-binding molecule of the present invention may further contain a dimerization domain. In certain embodiments, the dimerization domain may be present between the variable region of the heavy or light chain of the antibody and at least a part of the viral coat protein. This dimerization domain may contain at least one dimerization sequence and / or a sequence containing one or more cysteine residues. This dimerization domain may preferably be linked to the C-terminus of the heavy chain variable region or constant region. The dimerization domain can have various structures depending on whether the antibody variable region is prepared as a fusion polypeptide component with a viral coat protein component (without an amber stop codon after the dimerization domain), or whether the antibody variable region is prepared mainly without containing a viral coat protein component (for example, having an amber stop codon after the dimerization domain). When the antibody variable region is prepared mainly as a fusion polypeptide with a viral coat protein component, bivalent presentation is brought about by one or more disulfide bonds and / or a single dimerization sequence.

[0168] In this specification, the term "different in sequence from each other" in the description of a plurality of antigen-binding molecules with different sequences means that the sequences of individual antigen-binding molecules in the library are different from each other. That is, the number of different sequences in the library reflects the number of different independent clones of the sequences in the library, and may be referred to as the "library size". In a normal phage display library, it is from 10 6 to 10 12 and it is possible to expand the library size up to 10 14 by applying known techniques such as the ribosome display method. However, the actual number of phage particles used during the panning selection of a phage library is usually 10 to 10,000 times larger than the library size. This excess multiple, also called the "library equivalent number", represents that there can be 10 to 10,000 individual clones having the same amino acid sequence. Therefore, the term "different in sequence from each other" in the present invention means that the sequences of individual antigen-binding molecules in the library excluding the library equivalent number are different from each other, more specifically, there are from 10 6 to 10 14 molecules, preferably from 10 7 to 10 12 molecules, more preferably from 10 8 to 10 11 , particularly preferably from 10 8 to 10 10 antigen-binding molecules that are different in sequence from each other.

[0169] Furthermore, the term "mainly composed of" in the description of a library mainly composed of a plurality of antigen-binding molecules of the present invention reflects the number of antigen-binding molecules having different binding activities to the first and / or second antigens (or the binding activities of antigen-binding molecules to the first, second, and / or fourth antigens) among the number of different independent clones of the sequences in the library. Specifically, there are at least 10 antigen-binding molecules showing such binding activities in the library 4Preferably, the molecule is present. More preferably, the present invention provides a library in which at least 10 antigen-binding molecules exhibiting such binding activity are present. 5 More preferably, the present invention provides a library in which at least 10 antigen-binding molecules exhibiting such binding activity are present. 6 Even more preferably, the present invention provides a library in which at least 10 antigen-binding molecules exhibiting such binding activity are present. 7 Particularly preferably, the present invention provides a library in which at least 10 antigen-binding molecules exhibiting such binding activity are present. 8 Preferably, the present invention provides a library in which at least 10 antigen-binding molecules exhibiting such binding activity are present. In another expression, it can also be preferably expressed as the ratio of antigen-binding molecules having different binding activities (or the binding activities of antigen-binding molecules against the first, second, and / or fourth antigens) to the first and / or second antigens among the number of different independent clones of sequences in the library. Specifically, the present invention provides a library in which antigen-binding molecules exhibiting such binding activity are contained in an amount of 0.1% to 80%, preferably 0.5% to 60%, more preferably 1% to 40%, even more preferably 2% to 20%, and particularly preferably 4% to 10% of the number of different independent clones of sequences in the library. In the case of a fusion polypeptide, polynucleotide molecule, or vector, it can be expressed in the same manner as above, in terms of the number of molecules or the ratio in the whole molecule. Also, in the case of a virus, it can be expressed in the same manner as above, in terms of the number of virus individuals or the ratio in the whole individual.

[0170] The library of the present invention includes a plurality of antigen-binding molecules (or a plurality of antigen-binding molecules containing the variable regions of antibodies that can bind to three different antigens but cannot bind to the three antigens simultaneously) that can bind to a first antigen and a second antigen different from the first antigen, but do not bind to the first antigen and the second antigen simultaneously, and at least 1 molecule, 10 molecules, 100 molecules, 1000 molecules, 10 4 molecules, 10 5 molecules, 10 6 molecules, 10 7 molecules or 10 8It is preferable that the molecule exists. A plurality of antigen-binding molecules (or a plurality of antigen-binding molecules containing the variable region of an antibody that can bind to a third antigen different from the first antigen and the second antigen but cannot bind to the first antigen and the second antigen simultaneously) containing the variable region of an antibody that can bind to the first antigen and a second antigen different from the first antigen, but cannot bind to the first antigen and the second antigen simultaneously, are 10 of the number of independent clones with different sequences in the library -7 %, preferably 10 -6 %, more preferably 10 -5 %, 10 -4 %, 10 -3 %, 10 -2 %, 10 -1 %, to 1% is provided.

[0171] As one aspect of the "library mainly composed of a plurality of antigen-binding molecules with different sequences from each other" in the present invention, it is a library mainly composed of (i) variable regions of a plurality of antibodies with different sequences from each other, or antigen-binding molecules containing the variable region, or (ii) nucleic acids encoding the variable regions of a plurality of antibodies with different sequences from each other, or antigen-binding molecules containing the variable region.

[0172] Here, a plurality of antigen-binding molecules with different sequences from each other are antigen-binding molecules containing a variable region in which at least one amino acid modification has been introduced with respect to the template sequence of the library (for example, the variable region of an antibody). Preferred regions for amino acid modification include the heavy chain variable region and / or the light chain variable region. More preferably, regions exposed to the solvent and loop regions in the variable region are included. Among them, CDR1, CDR2, CDR3, FR3 regions, and loop regions are preferred. Specifically, Kabat numbering 31-35, 50-65, 71-74, 95-102 of the H chain variable region and Kabat numbering 24-34, 50-56, 89-97 of the L chain variable region are preferred, and Kabat numbering 31, 52a-61, 71-74, 97-101 of the H chain variable region and Kabat numbering 24-34, 51-56, 89-96 of the L chain variable region are more preferred.

[0173] The above antigen-binding molecule is not particularly limited as long as it contains the variable region of an antibody, and may be an antibody fragment such as Fv, Fab, Fab', or an antibody containing an Fc region.

[0174] As the amino acid to be modified, for example, among the variable regions of the antibody that binds to the first antigen or the second antigen, an amino acid that does not lose its binding to the antigen due to amino acid modification is selected. Alternatively, as the amino acid to be modified, among the variable regions of the antibody that binds to three different antigens, an amino acid that does not lose its binding to the three antigens due to amino acid modification is selected.

[0175] The amino acid modification of the present invention may be used alone or in combination of a plurality of them. When used in combination of a plurality of them, the number of combinations is not particularly limited. For example, it is 2 or more and 30 or less, preferably 2 or more and 25 or less, 2 or more and 22 or less, 2 or more and 20 or less, 2 or more and 15 or less, 2 or more and 10 or less, 2 or more and 5 or less, 2 or more and 3 or less. When combining a plurality of them, the amino acid modification may be added to only one of the heavy chain variable region or the light chain variable region of the antibody, or may be appropriately distributed and added to both the heavy chain variable region and the light chain variable region.

[0176] In addition, to modify amino acid residues, it also includes randomly modifying the amino acids in the variable region of an antibody that binds to the first antigen or the second antigen (or the variable region of an antibody that binds to the first, second, or fourth antigen), or inserting a peptide that is known to have binding activity to a desired antigen in advance into the above-mentioned region. In this way, from the antigen-binding molecules with modifications, a variable region that can bind to the first antigen and the second antigen but cannot bind to these antigens simultaneously (or a variable region that can bind to three different antigens but cannot bind to these three antigens simultaneously) is selected, and thus the antigen-binding molecule of the present invention can be obtained. Examples of peptides that are known to have binding activity to a desired antigen in advance include the peptides shown in Table 1 above.

[0177] Whether it is a variable region that can bind to the first antigen and the second antigen but cannot bind to these antigens simultaneously (or a variable region that can bind to three different antigens but cannot bind to these three antigens simultaneously), and further, when either one of the first antigen and the second antigen exists on the cell surface and the other exists alone, when both exist alone, or when both exist on the same cell surface, it can bind to both the first antigen and the second antigen simultaneously, but when they are expressed on different cells, whether it is a variable region that cannot bind simultaneously (or when any one or more of three different antigens exist alone rather than on the cell surface, or when any two or more of three different antigens exist on the same cell surface, it can bind to these antigens simultaneously, but when they are expressed on different cells, whether it is a variable region that cannot bind simultaneously) can be confirmed in the same way according to the above method.

[0178] As used herein, "phage display" is a technique for presenting a variant polypeptide as a protein fused to at least a portion of a coat protein on the surface of a phage, such as a filamentous phage. The utility of phage display lies in the ability to rapidly and efficiently select sequences that bind to a target antigen with high affinity from a large library of randomized protein variants. The display of peptide and protein libraries on phage has been used to screen millions of polypeptides for specific binding properties. Multivalent phage display methods have been used to present small random peptides and small proteins through fusion to gene III or gene VIII of filamentous phage (Wells and Lowman (Curr. Opin. Struct. Biol. (1992) 3, 355-362) and references cited therein). In monovalent phage display, a library of proteins or peptides is fused to gene III or a portion thereof and expressed at low levels in the presence of wild-type gene III protein such that phage particles display one or zero copies of the fusion protein. Since the avidity effect is reduced compared to multivalent phage, selection is based on the intrinsic ligand affinity and phagemid vectors are used, which simplify DNA manipulation (Lowman and Wells, Methods: A Companion to Methods in Enzymology (1991) 3, 205-216).

[0179] "Phagemid" is a plasmid vector having a copy of the bacterial origin of replication, such as ColE1, and the intergenic region of bacteriophage. Any known bacteriophage, such as filamentous bacteriophage and lambda-type bacteriophage, can be appropriately used for the phagemid. The plasmid usually also contains a selection marker for antibiotic resistance. DNA fragments cloned into these vectors can be propagated as plasmids. When the cells into which these vectors are introduced have all the genes necessary for the production of phage particles, the replication mode of the plasmid changes to rolling circle replication, generating a copy of one strand of the plasmid DNA and packaging phage particles. Phagemids can form infectious or non-infectious phage particles. This term includes phagemids containing a phage coat protein gene, or a fragment thereof, ligated to the gene of a heterologous polypeptide as a gene fusion such that the heterologous polypeptide is presented on the surface of the phage particle.

[0180] The term "phage vector" means a double-stranded replicative form of a bacteriophage containing a heterologous gene and capable of replication. The phage vector has a phage origin of replication that enables phage replication and phage particle formation. The phage is preferably a filamentous bacteriophage, such as M13, f1, fd, Pf3 phage or derivatives thereof, or a lambda-type phage, such as lambda, 21, phi80, phi81, 82, 424, 434, others or derivatives thereof.

[0181] "Oligonucleotide" is a short, single-stranded or double-stranded polydeoxynucleotide chemically synthesized by known methods (e.g., solid-phase methods such as phosphotriester, phosphite, or phosphoramidite chemistry using the methods described in EP266032, or methods through deoxynucleotide H-phosphonate intermediates described by Froeshler et al. (Nucl. Acids Res. (1986) 14, 5399-5407)). Other methods include the polymerase chain reaction and other autoligation methods described below, and oligonucleotide synthesis on solid supports. All of these methods are described by Engels et al. (Agnew. Chem. Int. Ed. Engl. (1989) 28, 716-734). These methods are used if all of the nucleic acid sequences of a gene are known, or if the sequence of a nucleic acid complementary to the coding strand is available. Alternatively, if the target amino acid sequence is known, possible nucleic acid sequences can be appropriately deduced using the known and preferred coding residues for each amino acid residue. Oligonucleotides can be purified by polyacrylamide gel or molecular sizing columns, or by precipitation methods.

[0182] The terms "fusion protein" and "fusion polypeptide" refer to a polypeptide having two moieties covalently linked to each other, each moiety being a polypeptide having different properties. These properties can be biological properties such as in vitro or in vivo activity. Also, these properties can be a single chemical or physical property, such as binding to a target antigen, catalysis of a reaction, etc. The two moieties can be directly linked by a single peptide bond, or through a peptide linker containing one or more amino acid residues. Usually, the two moieties and the linker are present in the same reading frame. Preferably, the two moieties of the polypeptide are obtained from different or distinct polypeptides.

[0183] The term "coat protein" refers to a protein of which at least a part is present on the surface of a virus particle. From a functional perspective, a coat protein is any protein that binds to a virus particle during the process of virus construction in a host cell and remains bound to it until the virus infects another host cell. A coat protein can be a major coat protein or a minor coat protein. A minor coat protein is a coat protein that is usually present in the outer shell of a virus, and preferably there are at least about 5, more preferably at least about 7, and more preferably at least about 10 or more copies of the protein per billion. The major coat protein can have dozens, hundreds, or thousands of copies per billion. Examples of major coat proteins include the p8 protein of filamentous phage.

[0184] As a non-limiting aspect of the present invention, six methods for preparing a library are exemplified as follows. 1. A method of inserting a peptide (this term is used to include polypeptides and proteins) that binds to a second antigen (or a peptide that binds to a second antigen and a peptide that binds to a fourth antigen) into an antigen-binding molecule that binds to a first antigen. 2. A method of preparing a library in which various amino acids appear at positions where loops in an antigen-binding molecule can be modified (extended) to be long, and obtaining an antigen-binding molecule having binding activity against any second antigen (or any second antigen and any fourth antigen) from the library using the binding activity from the library to the antigen as an index. 3. Identifying amino acids that maintain the binding activity with a first antigen using an antibody prepared by site-directed mutagenesis from an antigen-binding molecule known to bind to the first antigen in advance, and obtaining an antigen-binding molecule having binding activity against any second antigen (or any second antigen and any fourth antigen) from a library in which the identified amino acids appear, using the binding activity to the antigen as an index. 4. In the method of 3, further, an antibody library is prepared in which various amino acids appear at positions where loops in the antigen-binding molecule can be modified (extended) to a longer length, and an antigen-binding molecule having binding activity to an arbitrary second antigen (or an arbitrary second antigen and an arbitrary fourth antigen) is obtained from the library using the binding activity to the antigen as an index 5. In the method of 1, 2, 3, or 4, a method of modifying so that a sugar chain addition sequence (e.g., NxS, NxT, x is an amino acid other than P) appears and adding a sugar chain recognized by a sugar chain receptor (e.g., adding a high-mannose type sugar chain, which is recognized by a high-mannose receptor. It is known that a high-mannose type sugar chain can be obtained by adding kifunensine during antibody expression (MAbs. 2012 Jul-Aug;4(4):475-87)) 6. In the method of 1, 2, 3, or 4, Cys, Lys, or a non-natural amino acid is inserted or substituted at a loop site or a site where modification to various amino acids was possible, and a domain that binds to a second antigen (or a domain that binds to a second antigen and a domain that binds to a fourth antigen) is added by a covalent bond (a method represented by an antibody-drug conjugate, a method of covalently binding to Cys, Lys, or a non-natural amino acid (mAbs 6:1, 34-45; January / February 2014, WO2009 / 134891A2, Bioconjug Chem. 2014 Feb 19;25(2):351-61)) In the six library preparation methods exemplified above, the positions where amino acids in the antigen-binding molecule are substituted or the positions where peptides are inserted into the antigen-binding molecule are preferably in the Fab or variable region portion of the antigen-binding molecule. Preferred regions include regions exposed to the solvent and loop regions in the variable region. Among them, the CDR1, CDR2, CDR3, FR3 regions, and loop regions are preferred. Specifically, Kabat numbering 31-35, 50-65, 71-74, 95-102 of the heavy chain variable region and Kabat numbering 24-34, 50-56, 89-97 of the light chain variable region are preferred, and Kabat numbering 31, 52a-61, 71-74, 97-101 of the heavy chain variable region and Kabat numbering 24-34, 51-56, 89-96 of the light chain variable region are more preferred.

[0185] As one aspect of the method of inserting a peptide (or a peptide that binds to a second antigen and a peptide that binds to a fourth antigen) that binds to a second antigen into an antigen-binding molecule that binds to a first antigen described in the above method 1., a method of inserting G-CSF as exemplified in Angew Chem Int Ed Engl. 2013 Aug 5;52(32):8295-8 can also be mentioned. As another aspect, the inserted peptide can be obtained from a library presenting the peptide, but it is also possible to use the whole or a part of a naturally occurring protein.

[0186] As one aspect of the present invention, a method for producing a library is provided, which includes the following steps. The following steps (a) and (b): (a) Using the variable region sequence of an antibody that binds to a first antigen as the template sequence of the library, a step of identifying amino acid modifications that satisfy any one or more of the following (i) to (iii); (i) A modification that does not substantially change the binding ability to the first antigen; (ii) A modification that does not substantially change the ECM binding ability; and (iii) an insertion of a peptide consisting of 1 to 25 amino acids into the CDR1, CDR2, CDR3 or FR3 region of the heavy chain variable region or the light chain variable region; and (b) designing a library comprising a nucleic acid encoding the template sequence and nucleic acids encoding variable regions having different sequences from each other and having one or more amino acid modifications identified in step (a) in the template sequence A method for producing a library, comprising the above. By the production method, a library mainly composed of a plurality of antigen-binding molecules having different sequences from each other, wherein the antigen-binding region in the antigen-binding molecule is a variable region of an antibody that can bind to a first antigen and a second antigen different from the first antigen, but cannot bind to the first antigen and the second antigen simultaneously, and either one of the first antigen and the second antigen is CD3, and the other antigen is a molecule expressed on the surface of T cells or other immune cells. A library can be produced. Further, by the production method, a library mainly composed of a plurality of antigen-binding molecules having different sequences from each other, wherein the antigen-binding region in the antigen-binding molecule is a variable region of an antibody that can bind to three different antigens (a first antigen, a second antigen different from the first antigen, and a fourth antigen different from the first antigen and the second antigen), but cannot bind to the three antigens simultaneously, and any one of the three antigens is CD3, and the remaining two antigens are molecules expressed on the surface of T cells or other immune cells. A library can also be produced.

[0187] The "template sequence (of the library)" in the present invention refers to the amino acid sequence of an antibody (for example, the variable region sequence of an antibody, or the CDR sequence, etc.) that serves as the template array for constructing the library. By identifying modified amino acids that can be used for library construction using this sequence (for example, those that can be identified by the above aspects (a)(i), (ii), (iii)), a library of antigen-binding molecules can be constructed. For example, when the first antigen is CD3, those skilled in the art can appropriately select the variable region sequence of an antibody that binds to the CD3 antigen as the template sequence of the library.

[0188] In the above step (a)(i): In order to identify modified amino acids that do not substantially change the binding ability to CD3 (to identify amino acids that maintain the binding activity to CD3, which is the first antigen (in the case of human CD3, the γ-chain, δ-chain, or ε-chain that constitutes human CD3)), for example, amino acid modifications can be made at sites considered to be involved in antigen binding, and a single amino acid-modified antibody can be prepared and evaluated. For the evaluation of CD3 binding of the single amino acid-modified antibody, methods known to those skilled in the art can be appropriately selected. For example, it can be measured by ELISA, FACS (fluorescence activated cell sorting), ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), or the BIACORE method using the surface plasmon resonance (SPR) phenomenon.

[0189] In the above step (a)(i): To identify modified amino acids that do not substantially change the binding ability to CD3 (to identify amino acids that maintain the binding activity to CD3), for the antibody before modification, for example, the results of the ratio of the binding amounts of various variants can be used. That is, when the binding amount of the antibody before modification is X and the binding amount of the single amino acid modified variant is Y, the value of Z (the ratio of the binding amounts) = Y / X can be used. When Z (the ratio of the binding amounts) is 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, preferably 0.8 or more, it can be considered that the binding to the antibody before modification is maintained. An antibody library can be prepared so that these amino acids that maintain the binding appear.

[0190] ECM (Extracellular matrix) is one of the extracellular components and exists in various sites in the living body. Therefore, it is known that antibodies that strongly bind to ECM have poor blood kinetics (short half-life) (WO2012093704A1). Therefore, for the amino acids that appear in the antibody library, it is preferable to select amino acids that do not enhance ECM binding (to identify modified amino acids that do not substantially change the ECM binding ability).

[0191] In the above step (a)(ii): To identify modified amino acids that do not substantially change the ECM binding ability (to select amino acids that do not enhance ECM binding), for example, according to the method of Reference Example 2, evaluate the ECM binding, and use the value obtained by dividing the ECM binding value (ECL response value) of each variant by the ECM binding value of the antibody of MRA (heavy chain sequence number: 57, light chain sequence number: 58). This value can consider the effect of enhanced ECM binding by multiple modifications and can be adopted as valid up to 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, but preferably up to 10-fold can be adopted as valid in the library. An antibody library can be prepared so that the amino acids thus selected appear.

[0192] Note that, although not limited thereto, when the peptide insertion into CDR3 is 6 amino acids, since the binding to ECM is enhanced when there are many amino acids having a positive charge in the side chain in the extended loop of CDR3, it is preferable that no amino acid having 3 or more positive charges in the side chain appears in the loop.

[0193] In order to enhance the diversity of the library, the library of the present invention can identify (insert a peptide into the variable region) the modified amino acid inserted into the variable region in the above step (a)(iii). Preferable regions for peptide insertion include regions exposed to the solvent in the variable region and loop regions. Among them, CDR1, CDR2, CDR3, FR3 regions, and loop regions are preferable. Specifically, Kabat numbering 31-35, 50-65, 71-74, 95-102 of the H-chain variable region and Kabat numbering 24-34, 50-56, 89-97 of the L-chain variable region are preferable, and Kabat numbering 31, 52a-61, 71-74, 97-101 of the H-chain variable region and Kabat numbering 24-34, 51-56, 89-96 of the L-chain variable region are more preferable. Even more preferably, it is the region of Kabat numbering 99-100 of the H-chain variable region. Also, when modifying amino acids, amino acids that increase the binding activity to the antigen may be introduced together.

[0194] As a non-limiting aspect of the present invention, the length of the inserted peptide includes 1-3 amino acids, 4-6 amino acids, 7-9 amino acids, 10-12 amino acids, 13-15 amino acids, 15-20 amino acids, 21-25 amino acids, but preferably 1-3 amino acids, 4-6 amino acids, 7-9 amino acids.

[0195] To examine the insertion sites and lengths of peptides for enhancing library diversity, it can be carried out by preparing a molecule with a peptide inserted and evaluating the CD3 binding of the molecule. For the evaluation, methods known to those skilled in the art can be appropriately selected. For example, it can be measured by ELISA, FACS (fluorescence activated cell sorting), ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), or the BIACORE method using the surface plasmon resonance (SPR) phenomenon, etc.

[0196] As a non-limiting aspect of the present invention, an antibody library for obtaining an antibody that binds to CD3 and a second antigen (or CD3, a second antigen, and a fourth antigen) can be designed as follows. Step 1: Select amino acids that retain CD3 binding ability (the CD3 binding amount is 80% or more of the unmodified antibody) For example, a library for obtaining an antibody that binds to CD3 and a second antigen (or CD3, a second antigen, and a fourth antigen) can be prepared such that the amino acids selected in Step 1 appear.

[0197] As a non-limiting aspect of the present invention, an antibody library for obtaining an antibody that binds to CD3 and a second antigen (or CD3, a second antigen, and a fourth antigen) can be designed as follows. Step 1: Select amino acids that retain CD3 binding ability (the CD3 binding amount is 80% or more of the unmodified antibody) Step 2: Insert an amino acid between positions 99 - 100 (Kabat numbering) of the H-chain CDR3 For example, in addition to Step 1, by inserting an amino acid into the CDR3 region in Step 2, a library for obtaining an antibody that binds to CD3 and a second antigen (or CD3, a second antigen, and a fourth antigen) with enhanced library diversity can be prepared.

[0198] As a non-limiting aspect of the present invention, an antibody library for obtaining an antibody that binds to CD3 and a second antigen (or, CD3, a second antigen, and a fourth antigen) can be designed as follows. Step 1: Select amino acids that retain CD3-binding ability (the CD3-binding amount is 80% or more of the unmodified antibody). Step 2: Select amino acids for which ECM binding is within 10-fold compared to MRA before modification. Step 3: Insert an amino acid between positions 99-100 (Kabat numbering) of the H-chain CDR3. For example, in addition to Steps 1 and 3, by adding Step 2, it is possible to select amino acids for which ECM binding is not enhanced among the amino acids that appear in the library, but it is not limited to this method. Also, even in a library design that does not go through Step 2, it is possible to measure and evaluate ECM binding to the antigen-binding molecule obtained from the library.

[0199] As a non-limiting aspect of the present invention, when the VH region CE115HA000 (SEQ ID NO: 52) is used as the template sequence of the CD3 (CD3ε)-binding antibody, examples of the modified amino acids used in the library design include any one or more of the amino acids at positions 11, 31, 52a, 52b, 52c, 53, 54, 56, 57, 61, 72, 78, 98, 99, 100, 100a, 100b, 100c, 100d, 100e, 100f, 100g, 101 included in the heavy-chain variable region. The above library using the template sequence (SEQ ID NO: 94) with the amino acid modification of V11L / L78I added to the VH region CE115HA000 (SEQ ID NO: 52) is preferred, but not limited thereto. Further, the above library using the template sequence (SEQ ID NO: 95) with the amino acid modification of V11L / A52aD / L78I added to the VH region CE115HA000 (SEQ ID NO: 52), or the above library using the template sequence (SEQ ID NO: 96) with the CDR3 sequence extended is preferred, but not limited thereto. For example, when using the heavy chain variable region sequence described in SEQ ID NO: 96 as the template sequence, the modified amino acids used in the library design may include any one or more of the amino acids at positions 31, 52b, 52c, 53, 54, 56, 57, 61, 98, 99, 100, 100a, 100b, 100c, 100d, 100e, 100f, 100g in the Kabat numbering included in the heavy chain variable region. As another aspect, rCE115Hchain (SEQ ID NO: 97) can also be used as the template sequence of the CD3-binding antibody.

[0200] As a non-limiting aspect of the present invention, when using the VL region GLS3000 (SEQ ID NO: 53) as the template sequence of the CD3 (CD3ε)-binding antibody, the modified amino acids used in the library design may include any one or more of the amino acids at positions 24, 25, 26, 27, 27a, 27b, 27c, 27e, 30, 31, 33, 34, 51, 52, 53, 54, 55, 56, 74, 77, 89, 90, 92, 93, 94, 96 in the Kabat numbering included in the light chain variable region. As another aspect, rCE115Lchain (SEQ ID NO: 98) can also be used as the template sequence of the CD3-binding antibody.

[0201] As a non-limiting aspect of the present invention, a heavy chain variable region sequence containing heavy chain CDR1 (SEQ ID NO: 99), heavy chain CDR2 (SEQ ID NO: 100), and heavy chain CDR3 (SEQ ID NO: 101) can also be used as a template sequence for a CD3-binding antibody. As another aspect, a heavy chain CDR3 (SEQ ID NO: 102) with 6 amino acid residues inserted can also be used. Furthermore, as another aspect, a light chain variable region sequence containing light chain CDR1 (SEQ ID NO: 103), light chain CDR2 (SEQ ID NO: 104), and light chain CDR3 (SEQ ID NO: 105) can also be used as a template sequence for a CD3-binding antibody. As the amino acid sequence of the framework contained in the variable region, for example, the sequences of currently known fully human-type framework regions contained in websites such as IMGT (http: / / www.imgt.org / textes / IMGTrepertoire / ) can be appropriately used as the germline sequences contained in the antigen-binding molecules of the present invention, but it is not limited thereto.

[0202] Designing a library in the present invention (designing) includes, for example, using known library techniques such as NNK and TRIM Library (Gonzalez-Munoz A et al. MAbs 2012, Lee CV et al. J Mol Biol. 2004, Knappik A. et al. J Mol Biol. 2000, Tiller T et al. MAbs 2013) to design a library containing a plurality of variants of antigen-binding molecules containing variable regions in which the amino acids at specific sites are modified to the desired amino acids, but it is not particularly limited to this aspect.

[0203] "One or more amino acids" in the present invention is not particularly limited in the number of amino acids, and may be two or more types of amino acids, five or more types of amino acids, ten or more types of amino acids, fifteen or more types of amino acids, or twenty types of amino acids.

[0204] As one aspect of the present invention, there is provided a method for selecting a variable region with enhanced binding to a first antigen, which includes the following steps. The following steps (a) to (c): (a) A step of contacting the library of the present invention with a first antigen, (b) A step of recovering the antigen-binding molecules that bound to the first antigen in the step (a), and (c) A step of selecting, from the population of the antigen-binding molecules that bound to the first antigen in the step (b), an antigen-binding molecule containing a variable region with enhanced binding to the first antigen. A method for selecting a variable region with enhanced binding to a first antigen.

[0205] In this aspect, the first antigen is preferably CD3, but is not particularly limited.

[0206] The antigen-binding molecule used in the above selection method is not particularly limited as long as it contains the variable region of an antibody, and may be an antibody fragment such as Fv, Fab, Fab', or an antibody containing an Fc region.

[0207] In the above step (c), by measuring the binding to the first antigen by a method known to those skilled in the art, such as ELISA, FACS (fluorescence activated cell sorting), ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), or the BIACORE method using the surface plasmon resonance (SPR) phenomenon, an antigen-binding molecule containing a variable region with enhanced binding to the first antigen can be selected. Specifically, by comparing the binding activity of the antigen-binding molecule containing the variable region sequence selected as the template sequence of the library with the binding activity of the antigen-binding molecule that bound to the first antigen recovered in the step (b), when an antigen-binding molecule having a binding activity higher than the binding activity of the antigen-binding molecule containing the variable region sequence of the template is found, the antigen-binding molecule can be determined as an antigen-binding molecule containing a variable region with enhanced binding to the first antigen.

[0208] As the library used in the above step (a), a library produced by identifying amino acids retaining 80% or more of the CD3 binding ability is preferred, but it is not limited thereto.

[0209] As one aspect of the present invention, there is provided a method for producing an antigen-binding molecule, comprising the following steps. A method for producing an antigen-binding molecule comprising a variable region that can bind to a first antigen that is CD3 and a second antigen different from the first antigen, but does not bind to the first antigen and the second antigen simultaneously, comprising the following steps (a) to (c): (a) A step of contacting the library of the present invention with a second antigen; (b) A step of recovering the antigen-binding molecule bound to the second antigen in the step (a); and (c) A step of selecting, from the population of the antigen-binding molecules recovered in the step (b), an antigen-binding molecule comprising a variable region that does not bind to the first antigen and the second antigen simultaneously.

[0210] As another aspect of the present invention, there is provided a method for producing an antigen-binding molecule, comprising the following steps. A method for producing an antigen-binding molecule comprising a variable region that can bind to a first antigen that is CD3, a second antigen different from the first antigen, and a fourth antigen different from the first antigen and the second antigen, but does not bind to the three antigens simultaneously, comprising the following steps (a) to (f): (a) A step of contacting the library of the present invention with a second antigen; (b) A step of recovering the antigen-binding molecule bound to the second antigen in the step (a); (c) A step of selecting, from the population of the antigen-binding molecules recovered in the step (b), an antigen-binding molecule comprising a variable region that does not bind to the first antigen and the second antigen simultaneously; (d) A step of contacting the population of the antigen-binding molecules recovered in the step (c) with a fourth antigen; (e) A step of recovering the antigen-binding molecule bound to the fourth antigen in the step (d); and (f) Selecting, from the population of antigen-binding molecules recovered in step (e), an antigen-binding molecule comprising a variable region that does not bind simultaneously to the first antigen, the second antigen, and the fourth antigen.

[0211] In these embodiments, it is preferred that the first antigen is CD3, and the second antigen and the fourth antigen are molecules expressed on the surface of T cells or other immune cells. Further, it is preferred that the second antigen is an FcγR, TLR, lectin, IgA, immune checkpoint molecule, TNF superfamily molecule, TNFR superfamily molecule, or NK receptor molecule.

[0212] The antigen-binding molecule is not particularly limited as long as it contains the variable region of an antibody, and may be an antibody fragment such as Fv, Fab, Fab', or an antibody containing an Fc region.

[0213] In the present production method, step (c) may be the following selection step: (d) Selecting, from the population of antigen-binding molecules recovered in step (b), an antigen-binding molecule comprising a variable region that has binding activity to the first antigen and the second antigen but does not bind simultaneously to the first antigen and the second antigen expressed on different cells. In the present production method, step (f) may be the following selection step: (g) Selecting, from the population of antigen-binding molecules recovered in step (c), an antigen-binding molecule comprising a variable region that has binding activity to the first antigen, the second antigen, and the fourth antigen but does not bind simultaneously to the first antigen, the second antigen, and the fourth antigen expressed on different cells.

[0214] As one embodiment of the present invention, there is provided a method for producing a bispecific antibody comprising a common light chain variable region, the method including the following steps. A library mainly composed of a plurality of antigen-binding molecules with different arrays, wherein the antigen-binding regions in the antigen-binding molecules are variable regions consisting of a template sequence or variable regions having at least one amino acid modification in the template sequence, and can bind to a first antigen and a second antigen different from the first antigen, but are variable regions of an antibody that do not bind to the first antigen and the second antigen simultaneously. A method for producing a bispecific antibody, comprising the following steps (a) to (c): (a) A step of selecting the template sequence of the library of the present invention as a variable region that binds to a first antigen; (b) A step of selecting a variable region that binds to a second antigen but does not bind to the first antigen as a variable region that binds to the second antigen, comprising the following steps (i) to (iv); (i) A step of contacting the library of the present invention with a desired second antigen; (ii) A step of recovering the antigen-binding molecules bound to the second antigen in the step (i); (iii) A step of contacting the population of the antigen-binding molecules recovered in the step (ii) with the first antigen; (iv) A step of selecting the antigen-binding molecules that do not bind to the first antigen in the step (iii); and (c) A step of producing a bispecific antibody comprising the variable region that binds to the first antigen selected in the step (a) and the variable region that binds to the second antigen selected in the step (b).

[0215] In this aspect, it is preferable that the first antigen is CD3 and the second antigen is a molecule specifically expressed in cancer tissue.

[0216] By producing a bispecific antibody using this aspect, for example, it is also possible to efficiently obtain a common light chain variable region of a bispecific antibody, which includes a variable region that binds to CD3 as the first antigen and a variable region that binds to a molecule specifically expressed in cancer tissue as the second antigen.

[0217] The antigen-binding molecule used in the above step (b) is not particularly limited as long as it contains the variable region of an antibody, and may be an antibody fragment such as Fv, Fab, Fab', or an antibody containing an Fc region.

[0218] Regarding the presentation of the fusion polypeptide, the fusion polypeptide of the variable region of the antigen-binding molecule can be presented in various forms on the surface of cells, viruses, or phagemid particles. These forms include single-chain Fv fragments (scFv), F(ab) fragments, and multivalent forms of these fragments. The multivalent forms are preferably dimers of ScFv, Fab, or F(ab'), which are referred to herein as (ScFv)2, F(ab)2, and F(ab')2, respectively. One reason for the preference for the presentation of multivalent forms is that the presentation of multivalent forms enables the identification of clones that are usually of low affinity, or has multiple antigen-binding sites that enable more efficient selection of rare clones in the selection process.

[0219] Methods for presenting a fusion polypeptide containing an antibody fragment on the surface of bacteriophage are known in the art and are described, for example, in WO1992001047 and herein. Other related methods are described in WO1992020791, WO1993006213, WO1993011236, and 1993019172, and those skilled in the art can use these methods as appropriate. Other known literature (H.R. Hoogenboom & G. Winter (1992) J. Mol. Biol. 227, 381-388, WO1993006213, and WO1993011236) shows the identification of antibodies by an artificially rearranged variable region gene repertoire against various antigens presented on the phage surface.

[0220] When a vector is constructed for presentation in the form of scFv, the nucleic acid sequences encoding the variable region of the light chain and the variable region of the heavy chain of the antigen-binding molecule are included in this vector. Generally, the nucleic acid sequence encoding the variable region of the heavy chain of the antigen-binding molecule is fused to a viral coat protein component. The nucleic acid sequence encoding the variable region of the light chain of the antigen-binding molecule is linked to the variable region of the heavy chain of the antigen-binding molecule by a nucleic acid sequence encoding a peptide linker. The peptide linker generally contains about 5 to 15 amino acids. Optionally, other sequences encoding labels useful for, for example, purification or detection can be fused to the 3' end of the nucleic acid sequence encoding either or both the variable region of the light chain of the antigen-binding molecule or the variable region of the heavy chain of the antigen-binding molecule.

[0221] When a vector is constructed for presentation in the form of F(ab), the nucleic acid sequences encoding the variable region of the antigen-binding molecule and the constant region of the antigen-binding molecule are included in this vector. The nucleic acid encoding the variable region of the light chain is fused to the nucleic acid sequence encoding the constant region of the light chain. The nucleic acid sequence encoding the variable region of the heavy chain of the antigen-binding molecule is fused to the nucleic acid sequence encoding the constant CH1 region of the heavy chain. Generally, the nucleic acid sequences encoding the variable region and the constant region of the heavy chain are fused to the nucleic acid sequence encoding all or part of the viral coat protein. The variable region and the constant region of the heavy chain are preferably expressed as a fusion with at least part of the viral coat protein, and the variable region and the constant region of the light chain are expressed separately from the heavy chain viral coat fusion protein. The heavy chain and the light chain bind to each other, and the binding can be either covalent or non-covalent. Optionally, other sequences encoding polypeptide labels useful for, for example, purification or detection can be fused to either or both the 3' end of the nucleic acid sequence encoding the constant region of the light chain of the antigen-binding molecule or the 3' end of the nucleic acid sequence encoding the constant region of the heavy chain of the antigen-binding molecule.

[0222] Regarding the introduction of the vector into the host cell, the vector constructed as described above is introduced into the host cell for amplification and / or expression. The vector can be introduced into the host cell by known transformation methods including electroporation, calcium phosphate precipitation, etc. When the vector is an infectious particle such as a virus, the vector itself penetrates into the host cell. The fusion protein is presented on the surface of the phage particle by transfection of the host cell with a replicable expression vector into which the polynucleotide encoding the fusion protein is inserted and production of phage particles by known techniques.

[0223] The replicable expression vector can be introduced into the host cell using various methods. In one non-limiting embodiment, the vector can be introduced into the cell using the electroporation method as described in WO2000106717. The cells are optionally cultured at 37 °C in a standard culture medium for about 6 to 48 hours (or until the OD at 600 nm reaches 0.6 to 0.8), and then the culture supernatant is removed by centrifuging the culture (e.g., by decantation). At the initial stage of purification, the cell pellet is preferably resuspended in a buffer (e.g., 1.0 mM HEPES (pH 7.4)). The supernatant is then removed from the suspension by centrifugation again. The resulting cell pellet is resuspended in glycerol diluted, for example, to 5 - 20% V / V. The cell pellet is obtained by removing the supernatant from the suspension by centrifugation again. Based on the measured value of the cell concentration of the suspension obtained by resuspending the cell pellet in water or diluted glycerol, the final cell concentration is adjusted to the desired concentration using water or diluted glycerol.

[0224] For example, as a preferred recipient cell, Escherichia coli strain SS320 with electroporation response ability (Sidhu et al. (Methods Enzymol. (2000) 328, 333-363)) can be mentioned. Escherichia coli strain SS320 was prepared by conjugating MC1061 cells with XL1-BLUE cells under conditions sufficient to transfer a fertility episome (F' plasmid) or XL1-BLUE to MC1061 cells. The deposited number 98795 has been given to the Escherichia coli strain SS320 deposited at ATCC (10801 University Boulevard, Manassas, Virginia). Any F' episome that enables phage replication in this strain can be used in the present invention. Suitable episomes are available from strains deposited at ATCC or commercially available products are also available (TG1, CJ236, CSH18, DHF', ER2738, JM101, JM103, JM105, JM107, JM109, JM110, KS1000, XL1-BLUE, 71-18, etc.).

[0225] When using a higher DNA concentration (about 10 times) in electroporation, the transformation efficiency is improved and the amount of DNA transformed into the host cell increases. The use of a high cell density also enhances the efficiency (about 10 times). Due to the increase in the amount of transferred DNA, a library with greater diversity and a larger number of independent clones with different sequences can be prepared. Transformed cells are usually selected by the ability to grow on a medium containing an antibiotic.

[0226] Furthermore, the present invention provides a nucleic acid encoding the antigen-binding molecule of the present invention. The nucleic acid of the present invention may be in any form such as DNA, RNA, etc.

[0227] Furthermore, the present invention provides a vector containing the nucleic acid of the present invention. The type of vector can be appropriately selected by those skilled in the art according to the host cell into which the vector is introduced. For example, the above-mentioned vectors can be used.

[0228] Furthermore, the present invention relates to a host cell transformed with the vector of the present invention. The host cell can be appropriately selected by those skilled in the art. For example, the above-described host cells can be used.

[0229] In addition, the present invention provides a pharmaceutical composition comprising the antigen-binding molecule of the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention can be formulated by introducing a pharmaceutically acceptable carrier in addition to the antigen-binding molecule of the present invention by a known method. For example, it can be used parenterally in the form of a sterile solution with water or other pharmaceutically acceptable solutions, or an injection of a suspension. For example, it can be formulated by appropriately combining a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc., and mixing in a unit dosage form required for generally recognized pharmaceutical practice. Specifically, examples of carriers include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, calcium carmellose, sodium carmellose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinyl pyrrolidone, gelatin, medium-chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, etc. The amount of the active ingredient in these formulations is such that an appropriate volume within the indicated range is obtained.

[0230] The sterile composition for injection can be formulated according to ordinary pharmaceutical practice using a vehicle such as distilled water for injection. Examples of the aqueous solution for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, and appropriate solubilizing aids, such as alcohol, specifically ethanol, polyalcohols, such as propylene glycol, polyethylene glycol, and nonionic surfactants, such as polysorbate 80 (TM), HCO-50, may be used in combination.

[0231] Examples of the oily liquid include sesame oil and soybean oil, and they may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. Further, they may be formulated with a buffer, for example, phosphate buffer, sodium acetate buffer, a soothing agent, for example, procaine hydrochloride, a stabilizer, for example, benzyl alcohol, phenol, and an antioxidant. The prepared injection solution is usually filled into a suitable ampoule. Administration is preferably parenteral administration, and specifically, examples include injection dosage forms, nasal administration dosage forms, pulmonary administration dosage forms, transdermal administration forms, and the like. As an example of the injection dosage form, for example, it can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.

[0232] Also, the administration method can be appropriately selected according to the age and symptoms of the patient. As the dosage of the pharmaceutical composition containing the polypeptide or the polynucleotide encoding the polypeptide, for example, it can be selected in the range of 0.0001 mg to 1000 mg per kg of body weight per administration. Alternatively, for example, the dosage can be selected in the range of 0.001 to 100000 mg / body per patient, but these numerical values are not necessarily limiting. The dosage and administration method vary depending on the body weight, age, symptoms, etc. of the patient, but those skilled in the art can appropriately select them.

[0233] The present invention also provides a method for treating cancer, which includes the step of administering the antigen-binding molecule of the present invention, the antigen-binding molecule of the present invention for use in the treatment of cancer, the use of the antigen-binding molecule of the present invention in the manufacture of a therapeutic agent for cancer, and a process for manufacturing a therapeutic agent for cancer, which includes the step of using the antigen-binding molecule of the present invention.

[0234] The correspondence between the three-letter notation and the one-letter notation of amino acids used in this specification is as follows. Alanine: Ala: A; Arginine: Arg: R; Asparagine: Asn: N; Aspartic acid: Asp: D; Cysteine: Cys: C; Glutamine: Gln: Q; Glutamic acid: Glu: E; Glycine: Gly: G; Histidine: His: H; Isoleucine: Ile: I; Leucine: Leu: L; Lysine: Lys: K; Methionine: Met: M; Phenylalanine: Phe: F; Proline: Pro: P; Serine: Ser: S; Threonine: Thr: T; Tryptophan: Trp: W; Tyrosine: Tyr: Y; Valine: Val: V

[0235] It is naturally understood by those skilled in the art that any combination of one or more aspects described in this specification is also included in the present invention as long as it is not technically inconsistent based on the common general knowledge of those skilled in the art.

[0236] All prior art documents cited in this specification are incorporated herein by reference.

[0237] The present invention is further illustrated by the following examples, but is not limited to the following examples.

Example

[0238] 〔Example 1〕Concept of a modified immunoglobulin variable (Fab) region that binds to CD3 (the first antigen) and another antigen (the second antigen) and does not bind simultaneously to CD3 (the first antigen) and the other antigen (the second antigen) on different cells When an immunoglobulin binds simultaneously to two or more active FcγRs, or when it binds simultaneously to another antigen and an active FcγR, resulting in a cross-linking reaction of the active FcγR, the ITAM signal of the FcγR may be transmitted, potentially leading to the activation of immune cells. As described above, one molecule of an IgG-type antibody can bind to only one molecule of FcγR. Therefore, only in the presence of an antigen, two or more active FcγRs are cross-linked, leading to the activation of immune cells.

[0239] In addition, when an IgG-type antibody binds to an antigen in the variable region (Fab), it can simultaneously bind to one molecule of FcγR in the Fc region. Therefore, cross-linking occurs between the cell expressing the antigen and the FcγR-expressing cell. Depending on the cell expressing the antigen, there are cases where cross-linking between the antigen and FcγR is not preferred. Specifically, for example, when the antigen is CD3, there are cases where immunological activation such as cytokine release occurs when T cells cross-link with FcγR-expressing cells (J. Immunol. (1999) Aug 1, 163(3), 1246-52). In such cases, by introducing a modification into the Fc region, it is possible to eliminate the binding activity to FcγR and prevent the cross-linking reaction between the antigen and FcγR (Advanced Drug Delivery Reviews (2006) 58, 640-656). Similarly, when the antigen of an IgG-type antibody is a TNFR superfamily molecule such as CD40, OX40, CD27, or a TLR such as CD3 and TLR2, 4, 8, 9, etc., if cross-linking occurs via FcγR, immune activation occurs systemically, so it is not preferred to simultaneously bind to these molecules expressed on other cells.

[0240] On the other hand, although conventional multispecific antibodies can bind to multiple antigens simultaneously, depending on the combination of antigens, there are cases where it is not preferred to bind to multiple antigens simultaneously. For example, integrin αvβ3, known as an adhesion molecule, is expressed in many cancer cells and the blood vessels around tumors, and thus is useful as a target molecule for targeting tumors (R. Haubner, PLoS Med., 2, e70(2005)). However, on the other hand, it is known to be expressed in various normal cells (Thromb Haemost. 1998 Nov;80(5):726-34.). Therefore, if a multispecific antibody binds to both CD3 and integrin αvβ3 simultaneously, there is a possibility that normal cells will be damaged by the strong cytotoxic activity of T cells.

[0241] Therefore, as a method for controlling such an undesirable cross-linking reaction, in one variable (Fab) region, a variable region (Dual Binding Fab) that binds to a first antigen at a part thereof and binds to a second antigen at another part of the variable (Fab) region not involved in this binding was considered (Figure 1). At this time, as shown in Figure 1, when two adjacent parts in one variable (Fab) region are essential for binding to their respective antigens, binding of the first antigen inhibits binding of the second antigen, and similarly, binding of the second antigen inhibits binding of the first antigen. Therefore, since an improved antibody having such properties of Dual Binding Fab cannot bind to the first antigen and the second antigen simultaneously, it was considered that the cross-linking reaction between the first antigen and the second antigen does not occur (Figure 2). Also, when the first antigen and the second antigen are not expressed on the cell membrane like soluble proteins, or when both are present on the same cell, they can bind to both the first antigen and the second antigen simultaneously, but when they are expressed on different cells, they do not bind simultaneously and do not cross-link the two cells, which is also considered Dual Binding Fab (Figure 3). On the other hand, the antigen (third antigen) that binds to the other variable (Fab) region is considered to cause a cross-linking reaction with the first antigen (Figure 4) and also with the second antigen (Figure 5). As the constant region of the antibody, an Fc region that binds to FcγR can be used, or an Fc region with reduced binding activity to FcγR can also be used. By utilizing the properties of such Dual Binding Fab, for example, in a technique of damaging cancer cells expressing a cancer antigen by redirecting T cells via an antibody, it is possible to further enhance cancer specificity by imparting a targeting function to integrin in cancer tissue.

[0242] That is, if the variable (Fab) region can be improved to be Dual Binding Fab to endow the following properties, it is possible to create an antibody having the action as shown in Figure 1. 1. Having binding activity to the first antigen 2. Having binding activity against the second antigen 3. Not binding to the first antigen and the second antigen simultaneously Note that "not binding to the first antigen and the second antigen simultaneously" includes not crosslinking two cells, one expressing the first antigen and the other expressing the second antigen, or not binding to the first antigen and the second antigen expressed on separate cells simultaneously. Furthermore, when the first antigen and the second antigen are not expressed on the cell membrane like soluble proteins, or when both are present on the same cell, it is possible to bind to both the first antigen and the second antigen simultaneously, but when they are expressed on different cells, it also includes the case where they cannot bind simultaneously.

[0243] Similarly, if the variable (Fab) region is improved to obtain Dual Binding Fab and the following properties can be imparted, for example, it is possible to create an antibody having the action as shown in FIG. 6. 1. Having binding activity against the first antigen on T cells 2. Having binding activity against the second antigen on antigen-presenting cells 3. Not binding to the first antigen and the second antigen simultaneously

[0244] [Example 2] Preparation of anti-human and cynomolgus monkey CD3ε antibody CE115 (2-1) Preparation of hybridomas using rats immunized with human CD3 and cynomolgus monkey CD3-expressing cells SD rats (female, 6 weeks old at the start of immunization, Charles River Japan) were immunized with human CD3εγ or cynomolgus monkey CD3εγ-expressing Ba / F3 cells as follows. When the first immunization was set as day 0, on day 0, 5 x 10 7 human CD3εγ-expressing Ba / F3 cells were intraperitoneally administered together with Freund's complete adjuvant (Difco). On day 14, 5 x 10 7 cynomolgus monkey CD3εγ-expressing Ba / F3 cells were intraperitoneally administered together with Freund's incomplete adjuvant (Difco), and thereafter, 5 x 10 were administered 4 times every week 7Individual human or cynomolgus monkey CD3εγ-expressing Ba / F3 cells were alternately administered intraperitoneally. One week after the final administration of CD3εγ (day 49), human CD3εγ-expressing Ba / F3 cells were administered intravenously as a boost. Three days later, rat spleen cells and mouse myeloma cells SP2 / 0 were cell-fused according to a conventional method using PEG1500 (Roche Diagnostics). The fused cells, i.e., hybridomas, were cultured in RPMI1640 medium containing 10% FBS (hereinafter referred to as 10% FBS / RPMI1640).

[0245] On the day after fusion, (1) the fused cells were suspended in a semi-solid medium (StemCells), and selective culture of hybridomas was performed, and colony formation of hybridomas was carried out.

[0246] On the 9th or 10th day after fusion, hybridoma colonies were picked up and seeded at 1 colony per well in a 96-well plate containing HAT selection medium (10% FBS / RPMI1640, 2 vol% HAT 50x concentrate (Dainippon Pharmaceutical), 5 vol% BM-Condimed H1 (Roche Diagnostics)). After culturing for 3 to 4 days, the culture supernatant of each well was collected, and the concentration of rat IgG in the culture supernatant was measured. For the culture supernatant in which rat IgG was confirmed, clones producing antibodies specifically binding to human CD3εγ were selected by cell-ELISA with human CD3εγ-expressing Ba / F3 cells or Ba / F3 not expressing human CD3εγ attached (Figure 7). Furthermore, cross-reactivity to cynomolgus monkey CD3εγ was also evaluated by performing cell-ELISA with cynomolgus monkey CD3εγ-expressing Ba / F3 cells attached (Figure 7).

[0247] (2-2) Preparation of anti-human, cynomolgus monkey CD3ε chimeric antibody Total RNA was extracted from hybridoma cells using RNeasy Mini Kits (QIAGEN), and cDNA was synthesized using a SMART RACE cDNA Amplification Kit (BD Biosciences). Using the prepared cDNA, the variable region gene of the antibody was inserted into a cloning vector by PCR. The nucleotide sequences of each DNA fragment were determined using a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) with a DNA sequencer ABI PRISM 3700 DNA Sequencer (Applied Biosystems) according to the method described in the attached instructions. The CDRs and FRs of the CE115 H-chain variable region (SEQ ID NO: 13) and CE115 L-chain variable region (SEQ ID NO: 14) were determined according to Kabat numbering.

[0248] The chimeric antibody H-chain in which the above rat antibody H-chain variable region was combined with the human antibody IgG1 heavy chain constant region, and the chimeric antibody L-chain gene in which the above rat antibody L-chain variable region was combined with the human antibody kappa chain constant region were incorporated into an animal cell expression vector. Using the prepared expression vector, the CE115 chimeric antibody was expressed and purified (Reference Example 1).

[0249] (2-3) Preparation of EGFR_ERY22_CE115 Next, a molecule was prepared in which one Fab of an IgG with a cancer antigen (EGFR) as the basic backbone was replaced with a binding domain for CD3ε. At this time, as the Fc of the IgG serving as the basic backbone, a silent-type Fc with reduced binding affinity to FcgR (Fcγ receptor) was used as in the above-described case. As the binding domain for EGFR, Cetuximab-VH (SEQ ID NO: 15), which is the variable region of Cetuximab, and Cetuximab-VL (SEQ ID NO: 16) were used. As the antibody heavy chain constant region, G1d in which the C-terminal Gly and Lys of IgG1 were removed, A5 in which mutations of D356K and H435R were introduced into G1d, and B3 in which a mutation of K439E was introduced into G1d were used, and Cetuximab-VH-G1d (SEQ ID NO: 17), Cetuximab-VH-A5 (SEQ ID NO: 18), and Cetuximab-VH-B3 (SEQ ID NO: 19) combined with Cetuximab-VH were prepared according to the method of Reference Example 1. When the name of the antibody heavy chain constant region was designated as H1, the sequence corresponding to the heavy chain of the antibody having Cetuximab-VH in the variable region was shown as Cetuximab-VH-H1. Here, when indicating amino acid modifications, it was shown as D356K. The first alphabet (corresponding to D in D356K) means the alphabet when the amino acid residue before modification is represented by a single letter, the following number (corresponding to 356 in D356K) means the EU numbering of the modification site, and the last alphabet (corresponding to K in D356K) means the alphabet when the amino acid residue after modification is represented by a single letter.

[0250] EGFR_ERY22_CE115 (Figure 8) was prepared by replacing the VH domain and VL domain of the Fab against EGFR. That is, a series of expression vectors into which polynucleotides encoding EGFR ERY22_Hk (SEQ ID NO: 20), EGFR ERY22_L (SEQ ID NO: 21), CE115_ERY22_Hh (SEQ ID NO: 22), and CE115_ERY22_L (SEQ ID NO: 23) were inserted were prepared by a method known to those skilled in the art, such as the PCR method using primers to which appropriate sequences similar to the above-described method were added.

[0251] The expression vectors of the following combinations were introduced into FreeStyle293-F cells to transiently express each target molecule. · Target molecule: EGFR_ERY22_CE115 · Polypeptides encoded by the polynucleotides inserted into the expression vector: EGFR _ERY22_Hk, EGFR _ERY22_L, CE115_ERY22_Hh, CE115_ERY22_L

[0252] (2 - 4) Purification of EGFR_ERY22_CE115 The obtained culture supernatant was added to an Anti FLAG M2 column (Sigma). After washing the column, elution was performed with 0.1 mg / mL FLAG peptide (Sigma). The fraction containing the target molecule was added to a HisTrap HP column (GE Healthcare). After washing the column, elution was performed with a concentration gradient of imidazole. After the fraction containing the target molecule was concentrated by ultrafiltration, the fraction was added to a Superdex 200 column (GE Healthcare), and each purified target molecule was obtained by recovering only the monomer fraction of the eluate.

[0253] (2 - 5) Measurement of cytotoxic activity using human peripheral blood mononuclear cells (2 - 5 - 1) Preparation of human peripheral blood mononuclear cell (PBMC) solution Using a syringe pre-injected with 100 μL of a heparin solution at 1,000 units / mL (Novoparin injection, 5,000 units, Novo Nordisk), 50 mL of peripheral blood was collected from healthy adult volunteers. The peripheral blood, which had been diluted two-fold with PBS(-) and then divided into four equal parts, was added to a Leucosep lymphocyte separation tube (Cat. No. 227290, Greiner bio-one) into which 15 mL of Ficoll-Paque PLUS had been pre-injected and centrifuged. After centrifugation of the separation tube (2,150 rpm, 10 minutes, room temperature), the mononuclear cell fraction layer was collected. After washing the cells in the mononuclear cell fraction once with Dulbecco's Modified Eagle's Medium containing 10% FBS (SIGMA, hereinafter 10% FBS / D-MEM), the cells were prepared using 10% FBS / D-MEM so that the cell density became 4×10 6 / mL. The cell solution thus prepared was used as a human PBMC solution in subsequent tests.

[0254] (2-5-2) Measurement of cytotoxic activity Cytotoxic activity was evaluated by the cell growth inhibition rate using an xCELLigence real-time cell analyzer (Roche Diagnostics). As the target cells, the SK-pca13a cell line established by forced expression of human EGFR in the SK-HEP-1 cell line was used. SK-pca13a was detached from the dish and seeded at 1×10 4 cells / well at 100 μL / well in an E-Plate 96 plate (Roche Diagnostics), and measurement of viable cells was started using an xCELLigence real-time cell analyzer. The next day, the plate was taken out from the xCELLigence real-time cell analyzer, and 50 μL of each antibody prepared at each concentration (0.004, 0.04, 0.4, 4 nM) was added to the plate. After reacting at room temperature for 15 minutes, 50 μL of the human PBMC solution prepared in (2-5-1) (2×10 5Cells / well) were added, and live cell measurements were initiated by resetting the plate in the xCELLigence real-time cell analyzer. The reaction was carried out under conditions of 5% carbon dioxide and 37 °C, and the cell growth inhibition rate (%) was determined from the Cell Index value 72 hours after the addition of human PBMC using the following formula. The Cell Index value used in the calculation was the value after normalization so that the Cell Index value immediately before the addition of the antibody was 1. Cell growth inhibition rate (%) = (A - B) × 100 / (A - 1) A represents the average value of the Cell Index values in wells without the addition of the antibody (only target cells and human PBMC), and B represents the average value of the Cell Index values in each well. The test was performed in triplicate.

[0255] When measuring the cytotoxic activity of EGFR_ERY22_CE115 using CE115 with PBMC prepared from human blood as effector cells, extremely strong activity was observed (Figure 9).

[0256] Example 3 Preparation of antibodies that bind to CD3 and human integrin αvβ3 but do not bind simultaneously As shown in Figures 1 to 6, a Dual binding Fab is a molecule that binds to CD3 (the first antigen) and the target antigen (the second antigen) in the variable (Fab) region but does not bind to CD3 (the first antigen) and the target antigen (the second antigen) simultaneously. When introducing an amino acid modification into the Fab region of an antibody that binds to CD3 (the first antigen) to bind to the second antigen, usually amino acid modifications are introduced into both of the two H chains or L chains. However, when modifications are introduced into both the H chains or L chains, the two Fabs of the antibody bind to the two antigens respectively, and there is a possibility that the two Fabs will bind to CD3 (the first antigen) and the target antigen (the second antigen) simultaneously and crosslink. Therefore, one Fab of the antibody is made to be a Fab that binds to a third antigen or does not bind to anything, and the other Fab is made to be a dual binding Fab so that the crosslinking reaction of CD3 (the first antigen) and the target antigen (the second antigen) does not occur.

[0257] (3-1) Preparation of antibodies that bind to CD3 and human integrin αvβ3 but not simultaneously Integrin αvβ3, known as an adhesion molecule, is expressed in many cancer cells and blood vessels around tumors, and thus is useful as a target molecule for targeting tumors. On the other hand, it is known to be expressed in various normal cells (Thromb Haemost. 1998 Nov;80(5):726-34.). Therefore, if CD3 and integrin αvβ3 bind simultaneously, there is a possibility that normal cells will be damaged by the strong cytotoxic activity of T cells. Thus, it was thought that if a molecule that does not bind CD3 and integrin αvβ3 simultaneously could be prepared, an anti-EGFR antibody molecule could be targeted to tumor cells expressing integrin αvβ3 without damaging normal cells. That is, it was considered to obtain a dual binding Fab molecule that binds to EGFR with one variable region (Fab), binds to CD3, the first antigen, with another variable region, binds to integrin αvβ3, the second antigen, and does not bind to CD3 and integrin αvβ3 simultaneously.

[0258] If it can be shown that "a molecule in which the Fab region binds to CD3 under the condition that integrin αvβ3 is absent and the Fab region binds to integrin αvβ3 under the condition that CD3 is absent, and the molecule that binds to CD3 does not bind to integrin αvβ3, or the molecule that binds to integrin αvβ3 does not bind to CD3", it can be said that a dual binding Fab molecule having the characteristics of the target dual binding Fab (that is, it can bind to CD3 and the second antigen and does not bind to CD3 and the second antigen simultaneously) has been created.

[0259] (3-2) Acquisition of an antibody having a Fab region that binds to integrin αvβ3 As methods for obtaining dual-binding Fab molecules, a method using a library and a method of inserting a peptide known to have binding activity to a protein were considered. As a peptide having binding activity to integrin αvβ3, the RGD (Arg-Gly-Asp) peptide is known. Therefore, a heterodimeric antibody having one Fab as an EGFR binding domain and the other Fab as a CD3 binding domain and an integrin αvβ3 binding domain, which is an antibody that binds to CD3ε, CE115 (heavy chain variable region SEQ ID NO: 13, light chain variable region SEQ ID NO: 14), was prepared according to Reference Example 1 by inserting an RGD peptide into the loop portion of the heavy chain. That is, a series of expression vectors were prepared by inserting a polynucleotide encoding any of the following together with polynucleotides encoding EGFR ERY22_Hk (SEQ ID NO: 20), EGFR ERY22_L (SEQ ID NO: 21), and CE115_ERY22_L (SEQ ID NO: 23): · CE115_2 ERY22_Hh (SEQ ID NO: 24, substituting K and N for Kabat numberings 52b-53 respectively), · CE115_4 ERY22_Hh (SEQ ID NO: 25, substituting S and N for Kabat numberings 52b-54 respectively), · CE115_9 ERY22_Hh (SEQ ID NO: 26, inserting RGD between Kabat numberings 52a-52b), · CE115_10 ERY22_Hh (SEQ ID NO: 27, inserting RGD between Kabat numberings 52b-52c), · CE115_12 ERY22_Hh (SEQ ID NO: 28, inserting RGD between Kabat numberings 72-73), · CE115_17 ERY22_Hh (SEQ ID NO: 29, substituting K and S for Kabat numberings 52b-52c respectively), · CE115_47 ERY22_Hh (SEQ ID NO: 30, inserting RGD between Kabat numberings 98-99), · CE115_48 ERY22_Hh (SEQ ID NO: 31, with RGD inserted between Kabat numbering 99 - 100), · CE115_49 ERY22_Hh (SEQ ID NO: 32, with RGD inserted between Kabat numbering 100 - 100a). Also, as a control, an antibody (EH240 - Kn125 / EH240 - Hl076 / L73; SEQ ID NOs: 33 / 34 / 35) with an RGD (Arg - Gly - Asp) peptide inserted into the CH3 region of the antibody reported in J. Biotech, 155, 193 - 201, 2011 was prepared according to Reference Example 1. This molecule that binds to integrin αvβ3 via the CH3 region is considered to be able to bind to CD3 and integrin αvβ3 simultaneously.

[0260] (3-3) Confirmation of the binding between integrin αvβ3 and the antibody Whether a molecule with an RGD (Arg - Gly - Asp) peptide inserted into the Fab region binds to integrin αvβ3 was determined by electrochemiluminescence (ECL method). Specifically, biotin - anti human IgG Ab (Southern biotech) diluted with a TBS solution containing 0.1% BSA, 0.1 g / L calcium chloride, and 0.1 g / L magnesium chloride (referred to as the dilution (+) solution), an antibody solution prepared at 5 μg / mL or 1 μg / mL, and integrin αvβ3 with a sulfo - tag added (R&D Systems) were used in Nunc - Immuno TM MicroWell TMAdd 25 μL each to each well of 96-well round plates (Nunc), mix, and then incubate overnight at 4°C to form antibody-antigen complexes. Add 150 μL each of a TBS solution containing 0.5% BSA, 0.1 g / L calcium chloride, and 0.1 g / L magnesium chloride (denoted as blocking (+) solution) to each well of a streptavidin plate (MSD) and incubate overnight at 4°C. After removing the blocking solution, wash three times with 250 μL of a TBS solution containing 0.1 g / L calcium chloride and 0.1 g / L magnesium chloride (denoted as TBS(+) solution). Add 75 μL each of the antibody-antigen complex solution to each well and incubate at room temperature for 2 hours to bind biotin-anti human IgG Ab to the streptavidin plate. After removing the antibody-antigen complex solution, wash three times with TBS(+) solution, add 150 μL each of READ buffer (MSD) to each well, and detect the luminescence signal of the sulfo-tag with a Sector Imager 2400 (MSD).

[0261] The results are shown in Fig. 11. The parental antibody, EGFR ERY22_Hk / EGFR ERY22_L / CE115 ERY22_Hh / CE115_ERY22_L, showed no binding activity to integrin αvβ3, whereas binding to integrin αvβ3 was observed for EGFR ERY22_Hk / EGFR ERY22_L / CE115_2 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_4 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_9 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_10 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_12 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_17 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_47 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_48 ERY22_Hh / CE115_ERY22_L, and EGFR ERY22_Hk / EGFR ERY22_L / CE115_49 ERY22_Hh / CE115_ERY22_L.

[0262] (3-4) Confirmation of the binding between CD3 (CD3ε) and the antibody Next, it was determined by the ECL method whether the antibody that binds to the integrin αvβ3 in the Fab region prepared in the previous section retains the binding activity to CD3. Specifically, biotin-anti human IgG Ab (Southern biotech) diluted with a TBS solution containing 0.1% BSA (referred to as the dilution (-) solution), the antibody solution prepared at 5 μg / mL or 1 μg / mL, and the CD3ε homodimer protein added with a sulfo-tag were used in Nunc-Immuno TM MicroWell TMAdd 25 μL each to each well of 96-well round plates (Nunc), mix, and then incubate overnight at 4°C to form antibody-antigen complexes. Add 150 μL each of a TBS solution containing 0.5% BSA (referred to as blocking (-) solution) to each well of a streptavidin plate (MSD) and incubate overnight at 4°C. After removing the blocking solution, wash three times with 250 μL of TBS solution (-) solution. Add 75 μL each of the antibody-antigen complex solution to each well and incubate at room temperature for 2 hours to bind biotin-anti human IgG Ab to the streptavidin plate. After removing the antibody-antigen complex solution, wash three times with TBS (-) solution, add 150 μL each of READ buffer (MSD) to each well, and detect the luminescence signal of the sulfo-tag with a Sector Imager 2400 (MSD).

[0263] The results are shown in Fig. 12. In addition to the parental antibody EGFR ERY22_Hk / EGFR ERY22_L / CE115 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_2 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_4 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_9 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_10 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_12 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_17 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_47 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_48 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_49 ERY22_Hh / CE115_ERY22_L, all showed binding to CD3.

[0264] (3-5) Confirmation by ECL method that integrin αvβ3 and CD3 do not bind to the Fab region simultaneously From the results up to the previous item, a molecule having binding activity to integrin αvβ3 and binding activity to CD3 was obtained. Next, it was determined whether the Fab region prepared up to the previous item binds to CD3 (CD3ε) and integrin αvβ3 simultaneously.

[0265] When a molecule with an RGD (Arg-Gly-Asp) peptide inserted into the Fab region binds simultaneously to integrin αvβ3 and CD3, adding integrin αvβ3 and biotinylated CD3 to the antibody solution allows binding to both antigens, enabling detection by the ECL method. Specifically, human CD3ε homodimer protein with added biotin diluted in the dilution (+) solution, an antibody solution prepared at 10 μg / mL or 5 μg / mL, and integrin αvβ3 with a sulfo-tag (R&D Systems) were added 25 μL each to the wells of Nunc-Immuno TM MicroWell TM 96 well round plates (Nunc). After mixing, they were incubated overnight at 4°C to form antibody-antigen complexes. The blocking (+) solution was added 150 μL each to the wells of a streptavidin plate (MSD) and incubated overnight at 4°C. After removing the blocking solution, they were washed 3 times with 250 μL of TBS(+) solution containing 0.1 g / L calcium chloride and 0.1 g / L magnesium chloride. 75 μL of the antibody-antigen complex solution was added to each well and incubated at room temperature for 2 hours to bind biotin-anti human IgG Ab to the streptavidin plate. After removing the antibody-antigen complex solution, they were washed 3 times with TBS(+) solution, 150 μL of READ buffer (MSD) was added to each well, and the luminescence signal of the sulfo-tag was detected with a Sector Imager 2400 (MSD).

[0266] The results are shown in FIGS. 13 and 14. In the Fab region, EGFR ERY22_Hk / EGFR ERY22_L / CE115_2 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_12 ERY22_Hh / CE115_ERY22_L, and EGFR ERY22_Hk / EGFR ERY22_L / CE115_17 ERY22_Hh / CE115_ERY22_L, in which the RGD (Arg-Gly-Asp) peptide was inserted, simultaneously bound to integrin αvβ3 and CD3, and thus strong signals were detected in the ECL measurement. On the other hand, the signals were weak in EGFR ERY22_Hk / EGFR ERY22_L / CE115_9 ERY22_Hh / CE115_ERY22_L and EGFR ERY22_Hk / EGFR ERY22_L / CE115_48 ERY22_Hh / CE115_ERY22_L (FIG. 13). Also, almost no signals were detected in the ECL measurement for EGFR ERY22_Hk / EGFR ERY22_L / CE115_4 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_10 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_47 ERY22_Hh / CE115_ERY22_L, and EGFR ERY22_Hk / EGFR ERY22_L / CE115_49 ERY22_Hh / CE115_ERY22_L (FIG. 14). That is, it was suggested that these antibodies did not bind to integrin αvβ3 when binding to CD3.

[0267] (3-6) Consideration on the fact that integrin αvβ3 and CD3 do not bind to the Fab region simultaneously by ECL method From the above results, it was possible to create an antibody having the characteristics of a dual binding Fab molecule that binds to CD3 (CD3ε) and integrin αvβ3, respectively, in one Fab and does not bind to CD3 (CD3ε) and integrin αvβ3 simultaneously. In this example, for an antibody having a variable region that binds to CD3, which is the first antigen, an RGD peptide that binds to integrin αvβ3, which is the second antigen, was inserted into the Fab in the variable region to impart binding activity to the second antigen and obtain a molecule that does not bind to CD3 and the second antigen simultaneously. By the same method, a dual binding Fab molecule having binding activity to an arbitrary second antigen can be obtained by inserting a peptide having binding activity to a protein as exemplified in WO2006036834 into a loop in the Fab. In addition, a peptide having binding activity to a protein can be obtained by preparing a peptide library using methods known to those skilled in the art and selecting a peptide having the desired activity (Pasqualini R., Nature, 1996, 380 (6572): 364-6). Furthermore, it is considered possible to create a dual binding Fab molecule having binding activity to an arbitrary second antigen by using a library of antigen-binding molecules in which the loop in the Fab as described in Example 5 was modified (extended) to be long. Since the variable region for the first antigen can be obtained by various methods known to those skilled in the art, it can be said that by using such a library, it is possible to create a dual binding Fab molecule having binding activity to an arbitrary first antigen and an arbitrary second antigen and not being able to bind to the first antigen and the second antigen simultaneously.

[0268] From the above results, it was shown that EGFR ERY22_Hk / EGFR ERY22_L / CE115_4 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_10 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_47 ERY22_Hh / CE115_ERY22_L, and EGFR ERY22_Hk / EGFR ERY22_L / CE115_49 ERY22_Hh / CE115_ERY22_L bind to CD3 and integrin αvβ3 and do not bind to CD3 and integrin αvβ3 simultaneously. That is, EGFR ERY22_Hk / EGFR ERY22_L / CE115_4 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_10 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_47 ERY22_Hh / CE115_ERY22_L, and EGFR ERY22_Hk / EGFR ERY22_L / CE115_49 ERY22_Hh / CE115_ERY22_L are molecules having dual binding Fab, and it was revealed that it is possible to create such molecules.

[0269] [Example 4] Preparation of an antibody that binds to CD3 and human toll-like receptor 2 (TLR2) but does not bind simultaneously (4-1) Preparation of an antibody that binds to CD3 and human TLR2 but does not bind simultaneously TLR2, known as a pattern recognition receptor, is mainly expressed in immune cells such as macrophages, dendritic cells, and B cells, and is useful as a target molecule for activating immune cells. In addition, TLR2 is known to be expressed in normal cells other than immune cells, such as epithelial cells and endothelial cells. By simultaneously binding to a cancer antigen and CD3, T cells expressing CD3 in the tumor environment are recruited, and cancer cells are damaged by T cells. At the same time, it was considered that by simultaneously binding the cancer antigen and TLR2, immune cells expressing TLR2 in the tumor environment could also be recruited and activated. It is possible to more strongly activate T cells and induce acquired immunity because immune cells recruited by TLR2 can take up cancer cells damaged by T cells, process antigens, and present them to HLA to activate T cells. However, it was considered that if CD3 and TLR2 bind simultaneously, immune cells and normal cells may be damaged by the strong cytotoxic activity of T cells. Therefore, it was thought that if a molecule that does not bind CD3 and TLR2 simultaneously could be created, these cells could be recruited without damaging immune cells and normal cells expressing TLR2. That is, it was considered to obtain a dual binding Fab molecule that binds to EGFR in one variable region (Fab), binds to CD3, the first antigen, in another variable region, binds to TLR2, the second antigen, and does not bind to CD3 and TLR2 simultaneously.

[0270] If it can be shown that "it is a molecule in which the Fab region binds to CD3 under the condition that TLR2 is absent and the Fab region binds to TLR2 under the condition that CD3 is absent, and the molecule bound to CD3 does not bind to TLR2, or the molecule bound to TLR2 does not bind to CD3", it can be said that a dual binding Fab molecule having the characteristics of the target dual binding Fab (that is, it can bind to CD3 and the second antigen and does not bind to CD3 and the second antigen simultaneously) has been created.

[0271] (4-2) Acquisition of an antibody having a Fab region that binds to TLR2 As a peptide having binding activity to human TLR2, the RWGYHLRDRKYKGVRSHKGVPR peptide (SEQ ID NO: 36) is known. Therefore, a heterodimeric antibody having one Fab as an EGFR binding domain and the other Fab as a CD3 binding domain and a TLR2 binding domain, which is an antibody that binds to CD3ε, CE115 (heavy chain variable region SEQ ID NO: 13, light chain variable region SEQ ID NO: 14), was prepared according to Reference Example 1 by inserting a TRL2 binding peptide into the loop portion of the heavy chain. That is, a series of expression vectors were prepared by inserting a polynucleotide encoding any of the following together with polynucleotides encoding EGFR ERY22_Hk (SEQ ID NO: 20), EGFR ERY22_L (SEQ ID NO: 21), and CE115_ERY22_L (SEQ ID NO: 23): · CE115_DU21 ERY22_Hh (SEQ ID NO: 37, with a TRL2 binding peptide inserted between Kabat numberings 52b - 52c), · CE115_DU22 ERY22_Hh (SEQ ID NO: 38, with a TRL2 binding peptide inserted between Kabat numberings 52b - 52c), · CE115_DU26 ERY22_Hh (SEQ ID NO: 39, with a TRL2 binding peptide inserted between Kabat numberings 72 - 73), · CE115_DU27 ERY22_Hh (SEQ ID NO: 40, with a TRL2 binding peptide inserted between Kabat numberings 72 - 73). Also, as a control, an antibody with a TLR2 binding peptide added to the C - terminus of the CH3 region (CE115_ ERY22_DU42_Hh, SEQ ID NO: 41), and an antibody with a peptide having Cys residues at both ends of the TLR2 binding peptide added to the C - terminus of the CH3 region (CE115_ ERY22_DU43_Hh, SEQ ID NO: 42) were prepared according to Reference Example 1. This molecule that binds to TLR2 via the CH3 region is considered to be able to bind to CD3 and TLR2 simultaneously.

[0272] (4-3) Confirmation of the binding between TLR2 and the antibody Whether a molecule with a TLR2-binding peptide inserted into the Fab region binds to TLR2 was determined by the electrochemiluminescence method (ECL method). Specifically, biotin-anti human IgG Ab (Southern biotech) diluted with a TBS solution containing 0.1% BSA (referred to as the dilution (-) solution), an antibody solution prepared at 5 μg / mL or 1 μg / mL, and TLR2 (abnova) with a sulfo-tag added were added to each well of a Nunc-Immuno TM MicroWell TM 25 μL of each was added to each well of 96 well round plates (Nunc), mixed, and then incubated overnight at 4°C to form an antibody-antigen complex. A TBS solution containing 0.5% BSA (referred to as the blocking (-) solution) was added to each well of a streptavidin plate (MSD) at 150 μL each and incubated overnight at 4°C. After removing the blocking solution, it was washed 3 times with 250 μL of TBS (-) solution. 75 μL of the antibody-antigen complex solution was added to each well and incubated at room temperature for 2 hours to bind biotin-anti human IgG Ab to the streptavidin plate. After removing the antibody-antigen complex solution, it was washed 3 times with TBS (-) solution, 150 μL of READ buffer (MSD) was added to each well, and the luminescence signal of the sulfo-tag was detected with a Sector Imager 2400 (MSD).

[0273] The results are shown in Fig. 15. The parental antibody, EGFR ERY22_Hk / EGFR ERY22_L / CE115 ERY22_Hh / CE115_ERY22_L, showed no binding activity to TLR2, whereas binding to TLR2 was observed for EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU21 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU22 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU26 ERY22_Hh / CE115_ERY22_L, and EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU27 ERY22_Hh / CE115_ERY22_L.

[0274] (4-4) Confirmation of the binding between CD3 (CD3ε) and the antibody Next, it was determined by the ECL method whether the antibody that binds to the TLR2 Fab region prepared in the previous section retains the binding activity to CD3 (CD3ε). Specifically, biotin-anti human IgG Ab (Southern biotech) diluted with a TBS solution containing 0.1% BSA (referred to as the dilution (-) solution), the antibody solution prepared at 5 μg / mL or 1 μg / mL, and the CD3ε homodimer protein added with a sulfo-tag were added to Nunc-Immuno TM MicroWell TM25 μL of each was added to each well of 96-well round plates (Nunc), mixed, and then incubated overnight at 4°C to form antibody-antigen complexes. A TBS solution containing 0.5% BSA (referred to as blocking (-) solution) was added to each well of a streptavidin plate (MSD) at 150 μL per well and incubated overnight at 4°C. After removing the blocking solution, it was washed three times with 250 μL of TBS solution (-) solution. 75 μL of the antibody-antigen complex solution was added to each well and incubated at room temperature for 2 hours to bind biotin-anti human IgG Ab to the streptavidin plate. After removing the antibody-antigen complex solution, it was washed three times with TBS (-) solution, 150 μL of READ buffer (MSD) was added to each well, and the luminescence signal of sulfo-tag was detected with a Sector Imager 2400 (MSD).

[0275] The results are shown in Figure 16. In addition to the parental antibody EGFR ERY22_Hk / EGFR ERY22_L / CE115 ERY22_Hh / CE115_ERY22_L, binding to CD3 was observed for EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU21 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU22 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU26 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU27 ERY22_Hh / CE115_ERY22_L as well.

[0276] (4-5) Confirmation by ECL method that TLR2 and CD3 do not bind to the Fab region simultaneously From the results up to the previous paragraph, a molecule having binding activity to TLR2 and also having binding activity to CD3 was obtained. Next, it was determined whether the Fab region prepared up to the previous paragraph binds to CD3 and TLR2 simultaneously.

[0277] When a molecule with a TLR2-binding peptide inserted in the Fab region binds to TLR2 and CD3 simultaneously, adding TLR2 and biotinylated CD3 to the antibody solution allows binding to both antigens, enabling detection by the ECL method. Specifically, human CD3ε homodimer protein with added biotin diluted in the dilution (-) solution, the antibody solution prepared at 10 μg / mL or 5 μg / mL, and TLR2 (R&D Systems) with a sulfo-tag were added TM MicroWell TM 25 μL each was added to each well of 96 well round plates (Nunc), mixed, and then incubated overnight at 4°C to form an antibody-antigen complex. 150 μL of the blocking (-) solution was added to each well of the streptavidin plate (MSD) and incubated overnight at 4°C. After removing the blocking solution, it was washed three times with 250 μL of TBS (-) solution containing 0.1 g / L calcium chloride and 0.1 g / L magnesium chloride. 75 μL of the antibody-antigen complex solution was added to each well and incubated at room temperature for 2 hours to bind biotin-anti human IgG Ab to the streptavidin plate. After removing the antibody-antigen complex solution, it was washed three times with TBS (-) solution, 150 μL of READ buffer (MSD) was added to each well, and the luminescence signal of the sulfo-tag was detected with a Sector Imager 2400 (MSD).

[0278] The results are shown in Fig. 17. EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU42 ERY22_Hh / CE115_ERY22_L and EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU43 ERY22_Hh / CE115_ERY22_L, to which a TLR2-binding peptide was added in the CH3 region, showed strong signals in the ECL measurement by simultaneously binding to TLR2 and CD3. On the other hand, almost no signals were detected in the ECL measurement for EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU21 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU22 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU26 ERY22_Hh / CE115_ERY22_L, and EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU27 ERY22_Hh / CE115_ERY22_L. That is, it was suggested that these antibodies did not bind to TLR2 when binding to CD3.

[0279] (4-6) Consideration on the fact that TLR2 and CD3 do not bind to the Fab region simultaneously by ECL method From the above results, it was possible to create an antibody having the characteristics of a dual binding Fab molecule that binds to CD3 and TLR2 respectively in one Fab and does not bind to CD3 and TLR2 simultaneously. In this example, for an antibody having a variable region that binds to CD3, which is the first antigen, the RWGYHLRDRKYKGVRSHKGVPR peptide that binds to TLR2, which is the second antigen, was inserted into the Fab in the variable region, thereby imparting binding activity to the second antigen and obtaining a molecule that does not bind to CD3 and the second antigen simultaneously. In the same manner, by inserting a peptide having binding activity to a protein as exemplified in WO2006036834 into a loop in the Fab, a dual binding Fab molecule having binding activity to an arbitrary second antigen can be obtained. In addition, a peptide having binding activity to a protein can be obtained by preparing a peptide library using methods known to those skilled in the art and selecting a peptide having the desired activity (Pasqualini R., Nature, 1996, 380 (6572) :364-6)). Furthermore, it is considered possible to create a dual binding Fab molecule having binding activity to an arbitrary second antigen by using a library of antigen-binding molecules in which the loop in the Fab as described in Example 5 is modified (extended) to be long. Since the variable region for the first antigen can be obtained by various methods known to those skilled in the art, it can be said that by using such a library, it is possible to create a dual binding Fab molecule having binding activity to an arbitrary first antigen and an arbitrary second antigen and not being able to bind to the first antigen and the second antigen simultaneously.

[0280] From the above results, it was shown that EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU21 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU22 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU26 ERY22_Hh / CE115_ERY22_L, and EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU27 ERY22_Hh / CE115_ERY22_L bind to CD3 and TLR2 and do not bind to CD3 and TLR2 simultaneously. That is, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU21 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU22 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU26 ERY22_Hh / CE115_ERY22_L, and EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU27 ERY22_Hh / CE115_ERY22_L are molecules having dual binding Fab, and it was revealed that it is possible to create such molecules.

[0281] [Example 5] Antibody modification for the production of an antibody that binds to CD3 and a second antigen (5-1) Examination of the insertion site and length of a peptide capable of binding to a second antigen It was examined to obtain a dual binding Fab molecule that binds to a cancer antigen with one variable region (Fab) and binds to CD3, the first antigen, and further binds to a second antigen, and does not bind to CD3 and the second antigen simultaneously. A heterodimeric antibody having one Fab as an EGFR binding domain and the other Fab as a CD3 binding domain, and a heterodimeric antibody in which a GGS peptide was inserted into the loop portion of the heavy chain of CE115, an antibody that binds to CD3ε, was prepared according to Reference Example 1.

[0282] That is, between K52B and S52c in CDR2, EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE31 ERY22_Hh / CE115_ERY22_L with GGS inserted ((SEQ ID NO: 20 / 21 / 43 / 23), EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE32 ERY22_Hh / CE115_ERY22_L with GGSGGS peptide (SEQ ID NO: 90) inserted ((SEQ ID NO: 20 / 21 / 44 / 23), and EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE33 ERY22_Hh / CE115_ERY22_L with GGSGGSGGS peptide (SEQ ID NO: 91) inserted ((SEQ ID NO: 20 / 21 / 45 / 23) were prepared. Similarly, between D72 and D73, which is a loop region in Framework 3, EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE34 ERY22_Hh / CE115_ERY22_L with GGS inserted ((SEQ ID NO: 20 / 21 / 46 / 23), EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE35 ERY22_Hh / CE115_ERY22_L with GGSGGS peptide (SEQ ID NO: 90) inserted ((SEQ ID NO: 20 / 21 / 47 / 23), and EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE36 ERY22_Hh / CE115_ERY22_L with GGSGGSGGS peptide (SEQ ID NO: 91) inserted ((SEQ ID NO: 20 / 21 / 48 / 23) were prepared. Also, between A99 and Y100 in CDR3, EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE37 ERY22_Hh / CE115_ERY22_L with GGS inserted ((SEQ ID NO: 20 / 21 / 49 / 23), EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE38 ERY22_Hh / CE115_ERY22_L with GGSGGS peptide inserted ((SEQ ID NO: 20 / 21 / 50 / 23), and EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE39 ERY22_Hh / CE115_ERY22_L with GGSGGSGGS peptide inserted ((SEQ ID NO: 20 / 21 / 51 / 23) were prepared.

[0283] (5-2) Confirmation of the binding of the CE115 antibody inserted with the GGS peptide to CD3ε Using a Biacore T100, it was confirmed whether the various antibodies produced maintained their binding ability to CD3ε. A biotinylated CD3ε epitope peptide was bound to a CM5 chip via streptavidin, and the produced antibodies were flowed as analytes to analyze the binding affinity.

[0284] The results are shown in Table 2. The binding affinities of CE35, CE36, CE37, CE38, and CE39 to CD3ε were equivalent to those of the parental antibody CE115. This indicated that it was possible to insert a peptide that binds to a second antigen into these loops. Also, since the binding affinities of CE36 and CE39 inserted with GGSGGSGGS did not decrease, it was shown that peptide insertions of up to at least 9 amino acids at these positions did not affect the binding ability to CD3ε.

[0285]

Table 2

[0286] That is, it was shown that by using such a peptide-inserted CE115 to obtain an antibody that binds to a second antigen, it was possible to produce an antibody that can bind to CD3 and the second antigen but does not bind simultaneously. Here, the amino acid sequence of the peptide to be inserted or substituted is randomly modified according to known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. U.S.A. (1985) 82, 488-492)) and Overlap extension PCR, and the binding activities of each variant are compared according to the above method. By determining the insertion and substitution sites and the types and lengths of the amino acids that can still exhibit the desired activity even when the amino acid sequence is modified, a library can be created.

[0287] [Example 6] Library Design for Obtaining Antibodies that Bind to CD3 and a Second Antigen (6-1) Regarding an antibody library for obtaining antibodies that bind to CD3 and a second antigen (also referred to as a Dual Fab Library) As the first antigen, CD3 (CD3ε) is selected, and the following six methods are exemplified as ways to obtain antibodies that bind to CD3 (CD3ε) and any second antigen. 1. A method of inserting a peptide or polypeptide that binds to a second antigen into the Fab domain that binds to the first antigen (in addition to the peptide insertions shown in Examples 3 and 4, there is also a method of inserting G-CSF as exemplified in Angew Chem Int Ed Engl. 2013 Aug 5;52(32):8295-8). The binding peptides or polypeptides can be obtained from a library presenting the peptide or polypeptide, but it is also possible to utilize the whole or a part of a naturally occurring protein. 2. A method of creating an antibody library in which various amino acids appear at positions where the loops in the Fab can be modified (extended) as shown in Example 5, and obtaining a Fab having binding activity against any second antigen from the antibody library using the binding activity from the antibody library to the antigen as an index 3. Using an antibody prepared by site-directed mutagenesis from a Fab domain known to bind to CD3 in advance, identifying the amino acids that maintain the binding activity to CD3, and obtaining a Fab having binding activity against any second antigen from an antibody library in which the identified amino acids appear, using the binding activity from the antibody library to the antigen as an index 4. In the method of 3, further creating an antibody library in which various amino acids appear at positions where the loops in the Fab can be modified (extended), and obtaining a Fab having binding activity against any second antigen from the antibody library using the binding activity from the antibody library to the antigen as an index In the method of 5.1.2.3.4, it is modified so that a glycosylation sequence (for example, NxS, NxT, where x is an amino acid other than P) appears, and a method of adding a sugar chain recognized by a sugar chain receptor (for example, adding a high-mannose type sugar chain, which is recognized by a high-mannose receptor. It is known that a high-mannose type sugar chain can be obtained by adding kifunensine during antibody expression (MAbs. 2012 Jul-Aug;4(4):475-87)) In the method of 6.1.2.3.4, Cys, Lys or a non-natural amino acid is inserted or substituted at a loop site or a site where modification is possible to various amino acids, and a domain that binds to a second antigen (a polypeptide, a sugar chain, a nucleic acid represented by a TLR agonist) is added by covalent bonding (a method represented by an Antibody drug conjugate, a method of covalently bonding to Cys, Lys or a non-natural amino acid, described in mAbs 6:1, 34-45; January / February 2014, WO2009 / 134891A2, Bioconjug Chem. 2014 Feb 19;25(2):351-61) Using the above method, a Dual binding Fab that binds to a first antigen and a second antigen and does not bind to each other simultaneously can be obtained, and a domain that binds to an arbitrary third antigen (referred to as the other variable region and described in Example 1) can be combined by methods known to those skilled in the art, such as the common L chain, Cross mab, and Fab arm exchange method.

[0288] (6-2) Preparation of a single amino acid modified antibody of a CD3 (CD3ε) binding antibody using a site-specific mutagenesis method As the template sequences for the VH and VL regions of the CD3 (CD3ε)-binding antibody, CE115HA000 (SEQ ID NO: 52) and GLS3000 (SEQ ID NO: 53) were selected, respectively. Amino acid modifications were made according to Reference Example 1 at the sites considered to be involved in antigen binding. The constant region of the H chain was pE22Hh (a sequence obtained by modifying the CH1 and subsequent sequences of natural IgG1 with L234A, L235A, N297A, D356C, T366S, L368A, Y407V and deleting the C-terminal GK sequence and adding the DYKDDDDK sequence (SEQ ID NO: 89); SEQ ID NO: 54), and the kappa chain (SEQ ID NO: 55) was used as the constant region of the L chain. The modified sites are shown in Table 3. For the evaluation of CD3 (CD3ε) binding activity, the single amino acid modified antibody was obtained as a one-arm antibody (an antibody lacking one of the Fab domains of natural IgG). Specifically, in the case of the modification of the H chain, the modified H chain linked to the constant region pE22Hh and Kn010G3 (a sequence obtained by modifying the amino acid sequence at position 216 and subsequent positions of natural IgG1 with C220S, Y349C, T366W, H435R; SEQ ID NO: 56) and GLS3000 with the kappa chain linked to the 3' side were used. In the case of the modification of the L chain, a sequence with the kappa chain linked to the 3' side of the modified L chain and CE115HA000 and Kn010G3 with pE22Hh linked to the 3' side as the H chain were used for expression and purification in FreeStyle293 cells (using the method of Reference Example 1).

[0289] [Table 3]

[0290] (6-3) CD3 Binding Evaluation of Single Amino Acid Modified Antibody (6-2) The single amino acid variants constructed and expressed and purified were evaluated using a Biacore T200 (GE Healthcare). After appropriately immobilizing the CD3ε homodimer protein on a Sensor chip CM4 (GE Healthcare) by an amino coupling method, an antibody at an appropriate concentration was injected as an analyte and allowed to interact with the CD3ε homodimer protein on the sensor chip. Then, 10 mmol / L Glycine-HCl (pH 1.5) was injected to regenerate the sensor chip. The measurements were carried out at 25 °C, and HBS-EP+ (GE Healthcare) was used as the running buffer. The measured results were used to calculate the dissociation constant K D (M) using a single-cycle kinetics model (1:1 binding RI = 0) for the binding amount and the sensorgram obtained by measurement. Biacore T200 Evaluation Software (GE Healthcare) was used for calculating each parameter.

[0291] (6-3-1) Modification of the H chain Table 4 shows the results of the ratio of the binding amounts of various H chain variants to the antibody CE115HA000 before modification. That is, it is the value of Z (ratio of binding amounts) = Y / X when the binding amount of the antibody containing CE115HA000 is X and the binding amount of the H chain single amino acid variant is Y. At this time, as shown in Figure 18, when Z is less than 0.8, it is recognized that the binding amount is very small from the sensorgram, suggesting that the dissociation constant K D (M) may not be calculable. Next, the ratio of the dissociation constants K D (M) of various H chain variants to CE115HA000 (= KD value of CE115HA000 / KD value of the variant) is shown in Table 5. When Z shown in Table 4 is 0.8 or more, since it is considered that binding to the antibody CE115HA000 before modification is maintained, the antibody library designed to have these amino acids appear can become a Dual Fab Library.

[0292]

Table 4

[0293]

Table 5

[0294] (6-3-2) Modification of the L chain Table 6 shows the results of the ratio of the binding amounts of various L chain variants to GLS3000, which is the antibody before modification. That is, when the binding amount of the antibody containing GLS3000 is X and the binding amount of the L chain single amino acid variant is Y, Z (the ratio of the binding amounts) = Y / X. At this time, as shown in Fig. 18, when Z is less than 0.8, it is recognized that the binding amount is very small from the sensorgram, and there is a suggestion that the dissociation constant K D (M) may not be calculable. Next, Table 7 shows the ratio of the dissociation constants K D (M) of various L chain variants to GLS3000. When Z shown in Table 6 is 0.8 or more, it is considered that binding to GLS3000, which is the antibody before modification, is maintained. Therefore, the antibody library designed to have these amino acids appear can be a Dual Fab Library.

[0295]

Table 6

[0296]

Table 7

[0297] (6-4) Evaluation of the binding of single amino acid modified antibodies to ECM (Extracellular matrix) ECM (Extracellular matrix; extracellular matrix) is one of the extracellular components and exists in various parts of the living body. Therefore, it is known that antibodies that strongly bind to ECM have poor blood kinetics (short half-life) (WO2012093704A1). Therefore, it is preferable to select amino acids that do not enhance ECM binding among the amino acids that appear in the antibody library.

[0298] Each H-chain or L-chain variant was obtained by the method shown in (6-2). Next, ECM binding was evaluated according to the method of Reference Example 2. The ECM binding values (ECL response; values of ECL reaction) of each variant were divided by the ECM binding value of the antibody of MRA (H-chain sequence number: 57, L-chain sequence number: 58) within the same plate or on the same implementation date and are shown in Table 8 (H-chain) and Table 9 (L-chain). As shown in Tables 8 and 9, a tendency to enhance ECM binding was observed in some modifications. Among the values shown in Table 8 (H-chain) and Table 9 (L-chain), considering the effect of enhancing ECM binding by multiple modifications, up to 10-fold was considered effective and adopted in the Dual Fab Library.

[0299]

Table 8

[0300]

Table 9

[0301] (6-5) Examination of the insertion position and length of peptides for enhancing the diversity of the library In Example 5, it was shown that a peptide could be inserted at each position using a GGS sequence without losing binding to CD3 (CD3ε). In the Dual Fab Library as well, if loop extension becomes possible, a library containing a greater variety of molecules (also expressed as having high diversity) would be formed, and it was thought that it would become possible to obtain Fab domains that bind to a diverse range of second antigens. Therefore, since it was predicted that the binding activity would decrease with peptide insertion, V11L / D72A / L78I / D101Q modifications were added to the CE115HA000 sequence to increase the binding activity to CD3ε, and a molecule with a GGS linker inserted into the sequence ligated with pE22Hh was prepared, and CD3 binding was evaluated. The GGS sequence was inserted between positions 99 - 100 according to Kabat numbering. The antibody molecule was expressed as a one-arm antibody. Specifically, the above-described heavy chain containing a GGS linker, Kn010G3 (SEQ ID NO: 56), and the light chain, GLS3000 (SEQ ID NO: 53) and the Kappa sequence (SEQ ID NO: 55) were ligated, and expression and purification were performed according to Reference Example 1.

[0302] (6-6) Confirmation of binding of the CE115 antibody with inserted GGS peptide to CD3 Binding of the modified antibody with inserted GGS peptide to CD3ε was performed using Biacore according to the method described in Example 6. As a result, as shown in Table 10, it became clear that insertion of the GGS linker into the loop site was possible. In particular, it was possible to insert a GGS linker into the heavy chain CDR3 region, which is important for antigen binding, and binding to CD3ε was maintained with insertions of 3, 6, or 9 amino acids. In this study, a GGS linker was used, but it is thought that an antibody library in which various amino acids appear instead of GGS would also be acceptable.

[0303]

Table 10

[0304] (6-7) Examination of library insertion into heavy chain CDR3 using NNS bases (6-6) enables the insertion of 3, 6, or 9 amino acids using a GGS linker. By creating a library with the insertion of 3, 6, or 9 amino acids and using an antibody acquisition method represented by the ordinary Phage display method, it was considered possible to obtain an antibody that binds to the second antigen. Therefore, when the insertion into CDR3 is 6 amino acids, it was examined whether the binding to CD3 is retained even when various amino acids appear at the site where 6 amino acids are inserted, using NNS bases (where various amino acids appear). Since a decrease in binding activity was expected, primers were designed using NNS bases so that 6 amino acids would be inserted between 99-100 (Kabat numbering) in the CDR3 of the CE115HA340 sequence (SEQ ID NO: 59), which has a higher CD3ε binding activity than CE115HA000. The antibody molecule was expressed as a One arm antibody. Specifically, the sequence obtained by linking the aforementioned H chain including the aforementioned modification, Kn010G3 (SEQ ID NO: 56), the L chain GLS3000 (SEQ ID NO: 53), and the Kappa sequence (SEQ ID NO: 55) was adopted, and expression and purification were performed according to Reference Example 1. The binding of the obtained modified antibody was evaluated by the method described in (6-3). The results are shown in Table 11. It was clarified that the binding to CD3 (CD3ε) is retained even when various amino acids appear at the site where the amino acids were extended. Furthermore, Table 12 shows the results of evaluating whether non-specific binding is enhanced by the method shown in Reference Example 2. As a result, since the binding to ECM is enhanced when there are many amino acids with a positive charge in the side chain in the extended loop of CDR3, it was desired that no amino acids with 3 or more positive charges in the side chain appear in the loop.

[0305]

Table 11

[0306]

Table 12

[0307] (6-7) Design and construction of the Dual Fab Library From the studies described in Example 6, the antibody library (Dual Fab Library) for obtaining antibodies that bind to CD3 and a second antigen was designed as follows. Step 1: Select amino acids that retain the ability to bind CD3 (CD3ε) (the amount of CD3 binding is 80% or more of CE115HA000). Step 2: Select amino acids for which ECM binding is within 10-fold compared to MRA compared to before modification. Step 3: Insert 6 amino acids between positions 99-100 (Kabat numbering) of the H chain CDR3. Note that since the antigen-binding site of Fab is diversified by only Step 1, it can be a library for identifying antigen-binding molecules that bind to a second antigen. Also, since the antigen-binding site of Fab is diversified by only Steps 1 and 3, it can be a library for identifying antigen-binding molecules that bind to a second antigen. Even in a library design that does not go through Step 2, the ECM binding of the obtained molecules can be measured and evaluated.

[0308] From the above, the H chain of the Dual Fab Library diversified with the V11L / L78I mutation added to the FR (framework) of CE115HA000 as the CDR as shown in Table 13, and the L chain diversified with the CDR of GLS3000 as shown in Table 14. These antibody library fragments can be synthesized by DNA synthesis methods known to those skilled in the art. As the Dual Fab library, (1) a library in which the H chain was diversified as shown in Table 13 and the L chain was fixed to the original sequence GLS3000 or the L chain with enhanced CD3ε binding described in Example 6, (2) a library in which the H chain was fixed to the original sequence (CE115HA000) or the H chain with enhanced CD3ε binding described in Example 6 and the L chain was diversified as shown in Table 14, (3) a library in which the H chain was diversified as shown in Table 13 and the L chain was diversified as shown in Table 14 can be created. The H chain was entrusted to DNA2.0, a DNA synthesis company, with the library sequence diversified with the V11L / L78I mutation added to the FR (framework) of CE115HA000 as the CDR as shown in Table 13, and an antibody library fragment (DNA fragment) was obtained. The obtained antibody library fragment was amplified by the PCR method and inserted into a phagemid for phage display. At this time, GLS3000 was selected as the L chain. Furthermore, the constructed phagemid for phage display was introduced into Escherichia coli by the electroporation method, and Escherichia coli carrying the antibody library fragment was produced.

[0309]

Table 13

[0310]

Table 14

[0311] 〔Example 7〕 Acquisition of Fab domains that bind to CD3 and a second antigen (IL6R) from the Dual Fab Library (7-1) Acquisition of Fab domains that bind to human IL6R A Fab domain (antibody fragment) that binds to human IL6R was identified from the Dual Fab library designed and constructed in Example 6. As an antigen, biotin-labeled human IL6R was used to enrich antibody fragments having binding ability to human IL6R. Phage production was carried out from Escherichia coli retaining the constructed phagemid for phage display. The phage population precipitated by adding 2.5 M NaCl / 10% PEG to the culture broth of Escherichia coli in which phage production was carried out was diluted with TBS to obtain a phage library solution. Next, BSA was added to the phage library solution to a final concentration of 4% BSA. As a panning method, a panning method using an antigen immobilized on magnetic beads, which is a general method, was referred to (J. Immunol. Methods. (2008) 332 (1-2), 2-9, J. Immunol. Methods. (2001) 247 (1-2), 191-203, Biotechnol. Prog. (2002) 18 (2) 212-20, Mol. Cell Proteomics (2003) 2 (2), 61-9). As the magnetic beads, NeutrAvidin coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or Streptavidin coated beads (Dynabeads M-280 Streptavidin) were used.

[0312] Specifically, 250 pmol of biotin-labeled antigen was added to the prepared phage library solution, and the phage library solution was contacted with the antigen at room temperature for 60 minutes. Magnetic beads blocked with BSA were added, and the complex of the antigen and the phage was bound to the magnetic beads at room temperature for 15 minutes. The beads were washed three times with TBST (TBS containing 0.1% Tween 20, TBS was manufactured by TaKaRa), and then further washed twice with 1 mL of TBS. Thereafter, the beads to which 0.5 mL of 1 mg / mL trypsin was added were suspended at room temperature for 15 minutes, and then immediately the beads were separated using a magnetic stand, and the phage solution was recovered. The recovered phage solution was added to 10 mL of Escherichia coli strain ER2738 in the logarithmic growth phase (OD600 was 0.4 - 0.5). The phage was infected into the Escherichia coli by gently stirring and culturing the above Escherichia coli at 37°C for 1 hour. The infected Escherichia coli was seeded onto a 225 mm x 225 mm plate. Next, a phage library solution was prepared by recovering the phage from the culture solution of the seeded Escherichia coli. This cycle is called panning and was repeated multiple times. In addition, 40 pmol of biotin-labeled antigen was used in the second and subsequent panning. Also, in the fourth panning, the phage was concentrated using the binding affinity to CD3 as an index. Specifically, 250 pmol of biotin-labeled CD3ε peptide antigen (amino acid sequence SEQ ID NO: 60) was added to the prepared phage library solution, and the phage library was contacted with the antigen at room temperature for 60 minutes. Magnetic beads blocked with BSA were added, and the complex of the antigen and the phage was bound to the magnetic beads at room temperature for 15 minutes. The beads were washed with 1 mL of TBS containing 0.1% Tween 20 and TBS. The beads to which 0.5 mL of 1 mg / mL trypsin was added were suspended at room temperature for 15 minutes, and then immediately the beads were separated using a magnetic stand, and the phage solution was recovered. The phage recovered from the trypsin-treated phage solution was added to 10 mL of Escherichia coli strain ER2738 in the logarithmic growth phase (OD600 was 0.4 - 0.7). The phage was infected into the Escherichia coli by gently stirring and culturing the above Escherichia coli at 37°C for 1 hour.The infected Escherichia coli was seeded onto a 225 mm x 225 mm plate. Next, a phage library solution was recovered by recovering phages from the culture solution of the seeded Escherichia coli. Furthermore, in order to prevent multiple phages from infecting a single Escherichia coli, the phage solution diluted 100,000-fold again was used to infect Escherichia...

Claims

Claim 1 A method for screening an antigen-binding molecule comprising a variable region that can bind to a first antigen and a second antigen different from the first antigen but does not bind to the first antigen and the second antigen simultaneously, comprising the following steps (a) to (c): A screening method wherein either one of the first antigen and the second antigen is CD3 and the other antigen is an FcγR, TLR, lectin, IgA, immune checkpoint molecule, TNF superfamily molecule, TNFR superfamily molecule or NK receptor molecule: (a) A library mainly composed of a plurality of antigen-binding molecules having different sequences, wherein at least one amino acid modification is introduced into a template sequence that binds to the CD3 chain in each of the plurality of antigen-binding molecules, and the template sequence includes SEQ ID NO: 13, 52, 94, 96, 97, or 59 as the heavy chain variable domain, and the modification of the one amino acid includes insertion or substitution of an amino acid at at least one position selected from H chain: 31, 52b, 52c, 53, 54, 56, 57, 61, 98, 99, 100, 100a, 100b, 100c, 100d, 100e, 100f, 100g (Kabat numbering), contacting the library with the second antigen; (b) Recovering the antigen-binding molecules that bound to the second antigen in step (a); and (c) Selecting, from the population of antigen-binding molecules recovered in step (b), an antigen-binding molecule comprising a variable region that does not bind to the first antigen and the second antigen simultaneously. Claim 2 The screening method according to claim 1, further comprising the following steps: (d) Contacting the library with the first antigen; (e) Recovering the antigen-binding molecules that bound to the first antigen in step (d); and (f) Selecting, from the population of antigen-binding molecules that bound to the first antigen in step (e), an antigen-binding molecule comprising a variable region in which the binding to the first antigen is enhanced as compared to the template sequence. Claim 3 A library for screening antigen-binding molecules that bind to a first antigen and a second antigen different from the first antigen, mainly composed of a plurality of antigen-binding molecules with different sequences from each other, wherein the antigen-binding region in the antigen-binding molecule is a variable region of an antibody consisting of a template sequence or a variable region of an antibody having at least one amino acid modification in the template sequence. A method for producing a bispecific antibody, comprising the following steps (a) to (c): (a) Selecting the template sequence of the library as the variable region that binds to the first antigen; (b) A step of selecting a variable region that binds to the second antigen but does not bind to the first antigen as the variable region that binds to the second antigen, comprising the following steps (i) to (iv): (i) Contacting the library with the second antigen; (ii) Recovering the antigen-binding molecules bound to the second antigen in step (i); (iii) Contacting the population of antigen-binding molecules recovered in step (ii) with the first antigen; and (iv) Selecting the antigen-binding molecules that do not bind to the first antigen in step (iii); and (c) Producing a bispecific antibody comprising the variable region that binds to the first antigen selected in step (a) and the variable region that binds to the second antigen selected in step (b); wherein one of the first antigen and the second antigen is CD3, and the other antigen is FcγR, TLR, lectin, IgA, immune checkpoint molecule, TNF superfamily molecule, TNFR superfamily molecule or NK receptor molecule, wherein the antibody variable regions capable of binding to the first antigen and the second antigen include an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH), and include amino acid insertions and substitutions into the template sequence that binds to the first antigen, and the template sequence is the sequence described in SEQ ID NO: 13, 52, 94, 96, 97, or 59 as the heavy chain variable domain. Furthermore, here, the amino acid to be modified is at least one amino acid selected from H chain: 31, 52b, 52c, 53, 54, 56, 57, 61, 98, 99, 100, 100a, 100b, 100c, 100d, 100e, 100f, 100g (Kabat numbering).

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