Antigen-binding molecule containing a modified antibody variable region

Abstract

To provide an antibody that induces both of the cytotoxic activity caused by T cells and the cytotoxic activity caused by other cells in a cancer antigen-specific manner while avoiding side effects.SOLUTION: The present invention provides an antigen-binding molecule, comprising an antibody variable region that can bind to a first antibody and a second antibody different from the first antibody but does not bind to the first antibody and the second antibody at the same time, and a variable region that binds to a third antibody different from the first antibody and the second antibody.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention provides an antigen-binding molecule comprising a variable region of an antibody that can bind to two different antigens (a first antigen and a second antigen) but not to both antigens simultaneously, and a variable region of an antibody that binds to a third antigen different from these antigens; a pharmaceutical composition comprising the antigen-binding molecule; and a method for producing the same. [Background technology]

[0002] Antibodies are attracting attention as pharmaceuticals due to their high stability in plasma and low incidence of side effects (Nat. Biotechnol. (2005) 23, 1073-1078 (Non-Patent Literature 1) and Eur J Pharm Biopharm. (2005) 59 (3), 389-396 (Non-Patent Literature 2)). Antibodies not only bind to antigens and exhibit agonist and antagonist activity, but also induce cytotoxic activity (also called effector function) by effector cells, such as ADCC (Antibody-Dependent Cytotoxicity), ADCP (Antibody-Dependent Cell phagocytosis), and CDC (Complement-Dependent Cytotoxicity). In particular, antibodies of the IgG1 subclass exhibit effector function against cancer cells, leading to the development of numerous antibody drugs in the field of oncology.

[0003] For antibodies to express ADCC, ADCP, and CDC, it is essential that the antibody's Fc region binds to antibody receptors (FcγR) and various complement components present on effector cells such as NK cells and macrophages. In humans, the FcγR protein family has been reported to include isoforms FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb, and their allotypes have also been reported (Immunol. Lett. (2002) 82, 57-65 (Non-patent Literature 3)). Of these isoforms, FcγRIa, FcγRIIa, and FcγRIIIa have a domain called ITAM (Immunoreceptor Tyrosine-based Activation Motif) in their intracellular domain, which transmits activation signals. On the other hand, only FcγRIIb possesses a domain called ITIM (Immunoreceptor Tyrosine-based Inhibitory Motif) in its intracellular domain, which transmits inhibitory signals. Both FcγRs are known to transmit signals through crosslinking by immune complexes (Nat. Rev. Immunol. (2008) 8, 34-47 (Non-Patent Literature 4)). In fact, when an antibody exerts effector function on cancer cells, FcγRs on the effector cell membrane cluster at the Fc region of the antibody bound to the cancer cell membrane, and an activation signal is transmitted in the effector cell. As a result, a cytotoxic effect is exerted, but since the crosslinking of FcγRs is limited to effector cells located near the cancer cell, it indicates that immune activation occurs only locally within the cancer cell (Ann. Rev. Immunol. (1988). 6. 251-81 (Non-Patent Literature 5)).

[0004] Natural immunoglobulins bind to antigens in their variable region and to receptors such as FcγR, FcRn, FcαR, and FcεR, as well as complement, in their constant region. FcRn, one of the binding molecules that interacts with the Fc region of IgG, binds one molecule to each heavy chain of the antibody, so it has been reported that two molecules of FcRn bind to one molecule of IgG-type antibody. However, unlike FcRn, FcγR interacts with the hinge region and CH2 domain of the antibody, and only one molecule of FcγR binds to one molecule of IgG-type antibody (J. Bio. Chem., (20001) 276, 16469-16477). Furthermore, it has been shown that the binding of FcγR to the Fc region of the antibody is important due to several amino acid residues in the antibody's hinge region and CH2 domain, as well as the glycan attached to EU numbering 297th Asn bound to the CH2 domain (Chem. Immunol. (1997), 65, 88-110 (Non-Patent Literature 6), Eur. J. Immunol. (1993) 23, 1098-1104 (Non-Patent Literature 7), Immunol. (1995) 86, 319-324 (Non-Patent Literature 8)). Focusing on this binding site, various variants of the Fc region with different FcγR binding characteristics have been studied, and Fc region variants with higher binding activity to activated FcγR have been obtained (WO2000 / 042072 (Patent Literature 1), WO2006 / 019447 (Patent Literature 2)). For example, Lazar et al. succeeded in increasing the binding activity of human IgG1 to human FcγRIIIa (V158) by approximately 370 times by substituting Ser at EU number 239, Ala at EU number 330, and Ile at EU number 332 with Asn, Leu, and Glu, respectively (Proc. Natl. Acad. Sci. USA (2006) 103, 4005-4010 (Non-Patent Literature 9), WO2006 / 019447 (Patent Literature 2)). This modified strain has an A / I ratio of binding activity to FcγRIIIa and FcγIIb that is approximately 9 times higher than that of the wild type.Furthermore, Shinkawa et al. succeeded in increasing the binding activity to FcγRIIIa by approximately 100 times by deleting the fucose in the sugar chain attached to Asn at EU numbering 297 (J. Biol. Chem. (2003) 278, 3466-3473 (Non-Patent Literature 10)). These methods make it possible to significantly improve the ADCC activity of human IgG1 compared to natural human IgG1.

[0005] Conventional, naturally occurring IgG antibodies can only bind to one antigen because their variable region (Fab) allows them to recognize and bind to a single epitope. On the other hand, in cancer and inflammation, multiple proteins are known to be involved, and these proteins can crosstalk each other. For example, in immune diseases, several inflammatory cytokines (TNF, IL1, and IL6) are known to be involved (Nat. Biotech., (2011) 28, 502-10 (Non-Patent Literature 11)). Also, one mechanism by which cancer acquires drug resistance is the activation of other receptors (Endocr Relat Cancer (2006) 13, 45-51 (Non-Patent Literature 12)). In such cases, conventional antibodies that recognize a single epitope cannot inhibit multiple proteins.

[0006] As molecules that inhibit multiple targets, antibodies that bind to two or more antigens with a single molecule (called bispecific antibodies) are being studied. By modifying naturally occurring IgG-type antibodies, it is possible to confer binding activity to two different antigens (a first antigen and a second antigen) (MAbs. (2012) Mar 1, 4(2)). Therefore, in addition to neutralizing two or more antigens with a single molecule, they also have the effect of enhancing antitumor activity by cross-linking cells with cytotoxic activity with cancer cells. To date, various molecular forms of bispecific antibodies have been reported, including molecules with antigen-binding sites added to the N-terminus or C-terminus (DVD-Ig and scFv-IgG), molecules with two Fab regions having 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 region in the CH3 domain as a new antigen-binding site (Fcab) (Nat. Rev. (2010), 10, 301-316 (Non-Patent Literature 13), Peds (2010), 23(4), 289-297 (Non-Patent Literature 14)). Since all bispecific antibodies interact with FcγR in the Fc region, the effector function of the antibody is conserved. Therefore, for any antigen recognized by a bispecific antibody, it binds simultaneously with FcγR and exhibits ADCC activity against cells expressing the antigen.

[0007] If the antigens recognized by a bispecific antibody are all antigens specifically expressed in cancer cells, then binding to any of these antigens will exhibit cytotoxic activity against cancer cells, thus offering a more efficient anticancer effect than conventional antibody drugs that recognize only one antigen. However, if any one of the antigens recognized by the bispecific antibody is expressed in normal tissue or on immune cells, cross-linking with FcγR can cause damage to normal tissue and release cytokines (J. Immunol. (1999) Aug 1, 163(3), 1246-52 (Non-patent Literature 15)). As a result, severe side effects can be induced.

[0008] For example, Catumaxomab is known as a bispecific antibody that recognizes proteins expressed on T cells and proteins expressed on cancer cells (cancer antigens). Catumaxomab has two Fab molecules that bind to the cancer antigen (EpCAM) and the CD3ε chain expressed on T cells, respectively. Catumaxomab induces cytotoxic activity by T cells when the cancer antigen and CD3ε bind simultaneously, and induces cytotoxic activity by antigen-presenting cells such as NK cells and macrophages when the cancer antigen and FcγR bind simultaneously. By utilizing these two cytotoxic activities, Catumaxomab has shown high therapeutic efficacy in malignant ascites when administered intraperitoneally and is approved in Europe. (Cancer Treat Rev. (2010) Oct 36(6), 458-67 (Non-Patent Literature 16)) Furthermore, there have been reports of cases in which antibodies reacting to cancer cells appeared after Catumaxomab administration, revealing that adaptive immunity is induced (Future Oncol. (2012) Jan 8(1), 73-85 (Non-Patent Literature 17)). Based on these results, antibodies that possess both cytotoxic activity by T cells and action by cells such as NK cells and macrophages via FcγR (especially called trifunctional antibodies) are attracting attention because they are expected to have strong antitumor effects and induce adaptive immunity.

[0009] However, even in the absence of cancer antigens, trifunctional antibodies bind to both CD3ε and FcγR simultaneously. This allows T cells expressing CD3ε and cells expressing FcγR to cross-link even in environments without cancer cells, leading to the massive production of various cytokines. Due to this cancer antigen-independent induction of cytokine production, the administration of trifunctional antibodies is currently limited to intraperitoneal administration (Cancer Treat Rev. 2010 Oct 36(6), 458-67 (Non-Patent Literature 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 Literature 18)). Furthermore, with conventional bispecific antibodies, both the first antigen, the cancer antigen (EpCAM), and the second antigen, CD3ε, can bind to FcγR simultaneously. Therefore, it is structurally impossible to avoid such side effects caused by the simultaneous binding of FcγR and the second antigen, CD3ε.

[0010] In recent years, improved antibodies have been provided that utilize an Fc region with reduced binding activity to FcγR, thereby avoiding side effects while inducing cytotoxic activity by T cells (WO2012 / 073985). However, even such antibodies, due to their molecular structure, cannot bind to cancer antigens while simultaneously acting on the two immune receptors, CD3ε and FcγR. To date, no antibody has been known that can induce both T-cell-mediated cytotoxicity and non-T-cell-mediated cytotoxicity specifically in cancer antigens while avoiding side effects. [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] WO2000 / 042072 [Patent Document 2] WO2006 / 019447 [Non-patent literature]

[0012] [Non-Patent Document 1] Nat. Biotechnol. (2005) 23, 1073-1078 [Non-Patent Document 2] Eur J Pharm Biopharm. (2005) 59 (3), 389-396 [Non-Patent Document 3] Immunol. Lett. (2002) 82, 57-65 [Non-Patent Document 4] Nat. Rev. Immunol. (2008) 8, 34-47

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[0013] The present invention has been made in view of the above circumstances, and its objective is to provide an antigen-binding molecule comprising a variable region of an antibody in which one variable region 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 different from these antigens (a third antigen); a pharmaceutical composition comprising the antigen-binding molecule; and a method for producing the antigen-binding molecule. [Means for solving the problem]

[0014] The inventors diligently conducted research to solve the above problems. As a result, the inventors created an antigen-binding molecule that includes a variable region of an antibody in which one variable region 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 different from these antigens (a third antigen). By utilizing the binding activity of this antigen-binding molecule to three different antigens, they succeeded in enhancing the activity generated by the antigen-binding molecule. Furthermore, they succeeded in creating an antigen-binding molecule that can avoid crosslinking between different cells, which is thought to be a cause of side effects when conventional multispecific antigen-binding molecules are used as pharmaceuticals, by binding to antigens expressed on different cells.

[0015] More specifically, the present invention relates to the following: [1] 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 Variable region that binds to a third antigen different from the first and second antigens. Antigen-binding molecules, including those mentioned above. [2] An antigen-binding molecule comprising a variable region of an antibody in which the amino acids in 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. [3] The antigen-binding molecule according to [1] or [2], wherein the variable region that does not simultaneously bind to the first antigen and the second antigen is a variable region that does not simultaneously bind to the first antigen and the second antigen expressed on different cells. [4] Furthermore, an antigen-binding molecule according to any one of [1] to [3], which includes the Fc region of the antibody. [5] An antigen-binding molecule as described in [4], wherein the Fc region has reduced binding activity to FcγR compared to the Fc region of a natural human IgG1 antibody. [6] A multispecific antibody, which is an antigen-binding molecule as described in any of [1] to [5]. [7] An antigen-binding molecule according to any one of [1] to [6], wherein the variable region of the antibody capable of binding to a first antigen and a second antigen is a variable region in which at least one amino acid modification has been introduced. [8] The antigen-binding molecule according to [7], wherein the modification is the substitution or insertion of at least one amino acid. [9] The antigen-binding molecule according to [7] or [8], wherein the modification is the substitution of a portion of the amino acid sequence of a variable region that binds to a first antigen with an amino acid sequence that binds to a second antigen, or the insertion of an amino acid sequence that binds to a second antigen into the amino acid sequence of a variable region that binds to a first antigen.

[10] The antigen-binding molecule described in [8] or [9], wherein the number of inserted amino acids is 1 to 25.

[11] An antigen-binding molecule according to any one of [7] to

[10] , wherein the modified amino acid is an amino acid in the CDR1, CDR2, CDR3, or FR3 region of the variable region of the antibody.

[12] An antigen-binding molecule described in any of [7] to

[11] , wherein the amino acid to be modified is an amino acid in the loop region.

[13] An antigen-binding molecule according to any one of [7] to

[11] , wherein the modified amino acid is at least one amino acid selected from Kabat numbering 31-35, 50-65, 71-74 and 95-102 of the H chain variable region of the antibody, and Kabat numbering 24-34, 50-56 and 89-97 of the L chain variable region.

[14] An antigen-binding molecule according to any one of [1] to

[13] , wherein either the first antigen or the second antigen is a molecule specifically expressed on the surface of a T cell, and the other antigen is a molecule expressed on the surface of a T cell or other immune cell.

[15] The antigen-binding molecule described in

[14] , wherein either the first antigen or 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.

[16] The antigen-binding molecule described in

[14] or

[15] , wherein the third antigen is a molecule specifically expressed in cancer tissue. A pharmaceutical composition comprising an antigen-binding molecule described in any of [1] to

[16] and a medically acceptable carrier. A method for producing an antigen-binding molecule as described in any of

[18] [1] to

[16] , 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 a first antigen or a second antigen is modified, wherein at least one of the amino acids in the modified variable region comprises variable regions that are different from each other. (ii) A step of selecting from the prepared library an antigen-binding molecule that has binding activity to the first antigen and the second antigen, but includes a variable region that does not bind to the first antigen and the second antigen simultaneously. (iii) A step of culturing host cells containing a nucleic acid encoding the variable region of the antigen-binding molecule selected in step (ii), and / or a nucleic acid encoding the variable region of an antigen-binding molecule that binds to a third antigen, thereby expressing an antigen-binding molecule that includes a variable region of an antibody capable of binding to a first antigen and a second antigen, but to which the first and second antigens do not bind simultaneously, and / or a variable region that binds to a third antigen, and (iv) A step of recovering antigen-binding molecules from the host cell culture.

[19] The manufacturing method according to

[18] , wherein the variable region in the antigen-binding molecule selected in step (ii) 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 simultaneously, which are expressed on different cells.

[20] The method for producing the host cells cultured in step (iii) further comprises a nucleic acid encoding the Fc region of the antibody, as described in

[18] or

[19] .

[21] The manufacturing method according to

[20] , wherein the Fc region has reduced binding activity to FcγR compared to the Fc region of a natural human IgG1 antibody.

[22] The manufacturing method according to any one of

[18] to

[21] , wherein the antigen-binding molecule to be produced is a multispecific antibody.

[23] The manufacturing method according to any one of

[18] to

[22] , wherein at least one of the modified amino acids in the variable region in step (i) is a substituted or inserted amino acid.

[24] The manufacturing method according to

[23] , wherein the number of inserted amino acids is 1 to 25.

[25] The manufacturing method according to any one of

[18] to

[24] , 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.

[26] The manufacturing method according to any one of

[18] to

[25] , wherein the modification is a modification of an amino acid in the loop region.

[27] The manufacturing method according to any one of

[18] to

[25] , wherein the modification is a modification of at least one amino acid selected from Kabat numbering 31-35, 50-65, 71-74 and 95-102 of the H chain variable region of the antibody, and Kabat numbering 24-34, 50-56 and 89-97 of the L chain variable region.

[28] The method for producing a product according to any one of

[18] to

[27] , wherein either the first antigen or the second antigen is a molecule specifically expressed on the surface of a T cell, and the other antigen is a molecule expressed on the surface of a T cell or other immune cell.

[29] The method for producing the product according to

[28] , wherein either the first antigen or the second antigen is CD3, and the other antigen is FcγR, TLR, IgA, lectin, immune checkpoint molecule, TNF superfamily molecule, TNFR superfamily molecule, or NK receptor molecule.

[30] The method for producing a third antigen according to

[28] or

[29] , wherein the third antigen is a molecule specifically expressed in cancer tissue. A method for treating cancer, comprising the step of administering an antigen-binding molecule described in any of

[31] [1] to

[16] .

[32] An antigen-binding molecule according to any one of [1] to

[16] for use in the treatment of cancer.

[33] Use of an antigen-binding molecule described in any of [1] to

[16] in the manufacture of a cancer treatment agent. A process for manufacturing a cancer treatment agent, comprising the step of using an antigen-binding molecule as described in any of

[34] [1] to

[16] .

[35] It will be understood by those skilled in the art that any combination of one or more of the above-described embodiments is included in the present invention, insofar as it does not contradict the technical common sense of those skilled in the art. [Brief explanation of the drawing]

[0016] [Figure 1] This is a conceptual diagram of an antibody that binds to a first antigen and a second antigen, but not simultaneously. [Figure 2] This is a conceptual diagram of an antibody that does not cause crosslinking because it does not simultaneously bind to two antigens. [Figure 3] This is a conceptual diagram of an antibody that binds to two antigens simultaneously but does not bind to two cells simultaneously. [Figure 4] This is a conceptual diagram of an antibody that cross-links cancer cells with T cells expressing the primary receptor. [Figure 5] This is a conceptual diagram of an antibody that cross-links cancer cells with cells expressing a second receptor. [Figure 6] This is a conceptual diagram of an antibody that cross-links cancer cells and immune cells, but does not cross-link immune cells with each other. [Figure 7] This graph shows the results of cell ELISA for CE115 against CD3ε. [Figure 8] This is a diagram showing the molecular shape of EGFR_ERY22_CE115. [Figure 9] This graph shows the TDCC results (SK-pca13a) for EGFR_ERY22_CE115. [Figure 10] This graph shows the binding affinity of humanized CE115 to CD3ε. [Figure 11] This graph shows the results of an ECL-ELISA that detects the binding of RGD-inserted CE115 to integrins. [Figure 12] This graph shows the results of ECL-ELISA, which detects the binding of RGD-inserted CE115 to CD3ε. [Figure 13] This graph shows the results of ECL-ELSIA, which detects the simultaneous binding of RGD insertion CE115 to integrins and CD3ε. The results for the variant that exhibits simultaneous binding are shown. [Figure 14] This graph shows the results of ECL-ELISA for the simultaneous binding of RGD-inserted CE115. Results for the variant without simultaneous binding are also shown. [Figure 15] This graph shows the results of an ECL-ELISA that detects the binding of TLR2-binding peptide insertion CE115 to TLR2. [Figure 16]This graph shows the results of ECL-ELISA, which detects the binding of TLR2-binding peptide insertion CE115 to CD3ε. [Figure 17] This graph shows the results of an ECL-ELISA that detects the simultaneous binding of TLR2-binding peptide insertion CE115 to TLR2 and CD3. [Figure 18] This is an example of a sensorgram of an antibody with a binding ratio of less than 0.8. The vertical axis represents the RU value (response), and the horizontal axis represents time. [Figure 19] This figure shows the binding of the Fab domain presented by the phage to CD3ε and IL6R. [Figure 20] This figure shows the binding of the Fab domain presented by the phage to CD3ε and human IgA (hIgA). NC indicates binding to a plate without antigen immobilization. [Figure 21] This figure shows the binding of IgGized clone CD3ε to human IgA (hIgA). [Figure 22] This figure shows that the binding of IgG-modified clones to human IgA is inhibited by CD3ε, and that these clones cannot bind to human IgA (hIgA) and CD3ε simultaneously. [Figure 23] This figure shows the binding of the Fab domain presented by the phage to CD3ε and human CD154. NC indicates binding to a plate without antigen immobilization. [Figure 24] This figure shows the binding of IgG-modified clone CD3ε to human CD154. [Figure 25] This figure shows that the binding of IgG-modified clones to human CD154 is inhibited by CD3ε, and that these clones cannot bind to human CD154 and CD3ε simultaneously. [Modes for carrying out the invention]

[0017] In the present invention, the "variable region of an antibody" usually refers to a region composed of four framework regions (FRs) and three complementarity-determining regions (CDRs) sandwiched between them, and also includes partial sequences as long as they have activity to bind to part or all of an antigen. In particular, regions including antibody light chain variable regions (VLs) and antibody heavy chain variable regions (VHs) are preferred. The variable region of an antibody in the present invention may be any sequence, and may be the variable region of an antibody of any origin, such as mouse antibodies, rat antibodies, rabbit antibodies, goat antibodies, camel antibodies, and humanized antibodies derived from these non-human antibodies, as well as human antibodies. A "humanized antibody" is also called a reshaped human antibody, and is an antibody derived from a mammal other than a human, such as a mouse antibody, in which the complementarity-determining region (CDR) has been transplanted into the CDR of a human antibody. Methods for identifying CDRs are publicly known (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al., Nature (1989) 342: 877). General genetic recombination techniques are also publicly known (see European Patent Application Publication No. EP 125023 and WO 96 / 02576).

[0018] The statement that the "variable region of the antibody" of the present invention "does not bind to the first antigen and the second antigen simultaneously" 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. Here, "does not bind to the first antigen and the second antigen simultaneously" also includes cases where the antibody does not cross-link two cells, one expressing the first antigen and the other expressing the second antigen, or where it does not simultaneously bind to the first and second antigens expressed on separate cells. Furthermore, if the first and second antigens are not expressed on the cell membrane, like soluble proteins, or if both are present on the same cell, the antibody can simultaneously bind to both the first and second antigens, but if they are expressed on different cells, it cannot bind to both simultaneously. Such variable regions of antibodies are not particularly limited as long as they possess the desired function, but examples include variable regions in which some amino acids in the variable region of an IgG-type antibody are modified to bind to a desired antigen. For example, amino acids are selected from the variable region of an antibody that binds to a first or second antigen, and the modification does not result in the loss of binding to the antigen. Here, "expression on different cells" simply means that the expression is expressed on separate cells. Such cell combinations could be of the same type, such as a T cell and another T cell, or of different types, such as a T cell and an NK cell.

[0019] The amino acid modifications of the present invention may be used individually or in combination. When using multiple units in combination, the number of units to be combined is not particularly limited and can be set appropriately within the range that achieves the purpose of the invention. For example, 2 to 30 units, preferably 2 to 25 units, 2 to 22 units, 2 to 20 units, 2 to 15 units, 2 to 10 units, 2 to 5 units, or 2 to 3 units. When combining multiple modifications, the amino acid modification may be applied only to the heavy chain variable region or the light chain variable region of the antibody, or it may be applied to both the heavy chain variable region and the light chain variable region as appropriate.

[0020] Modification of one or more amino acid residues in the variable region is permitted, as long as the antigen-binding activity is maintained. When modifying amino acids in the variable region, it is preferable, though not particularly limited, that the binding activity of the antibody before modification is maintained. For example, it is preferable that the binding activity is 50% or more, preferably 80% or more, and more preferably 100% or more compared to the original antibody. Alternatively, the binding activity may increase due to the amino acid modification; for example, the binding activity may be 2 times, 5 times, 10 times, etc., compared to the original antibody.

[0021] Preferred regions for amino acid modification include the solvent-exposed regions and loop regions within the variable regions. Of these, the CDR1, CDR2, CDR3, FR3 regions and loop regions are preferred. Specifically, Kabat numberings 31-35, 50-65, 71-74, and 95-102 in the H chain variable region and Kabat numberings 24-34, 50-56, and 89-97 in the L chain variable region are preferred, with Kabat numberings 31, 52a-61, 71-74, and 97-101 in the H chain variable region and Kabat numberings 24-34, 51-56, and 89-96 in the L chain variable region being more preferred. In addition, amino acids that increase antigen binding activity may be introduced during amino acid modification.

[0022] In this invention, the term "loop region" refers to a region containing residues that are not involved in maintaining the β-barrel structure of immunoglobulin. In this invention, "modification of an amino acid" means any substitution, deletion, addition, insertion, or modification, or a combination thereof. In this invention, "modification of an amino acid" can be rephrased as "mutation of an amino acid," and both terms are used interchangeably.

[0023] When substituting amino acid residues, the aim is to modify aspects such as (a) to (c) below by substituting with other amino acid residues: (a) the backbone structure of the polypeptide in the sheet or helical region; (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 general side-chain properties: (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 chain orientation: gly, pro; and (6) Aromatic: trp, tyr, phe.

[0024] Substitutions of amino acid residues within each of these groups are called conservative substitutions, while substitutions of amino acid residues between different groups are called non-conservative substitutions. The substitution in this invention may be a conservative substitution, a non-conservative substitution, or a combination of a conservative substitution and a non-conservative substitution.

[0025] Furthermore, modifications to amino acid residues include selecting a variable region of an antibody that binds to either the first or second antigen, from among those randomly modified to include amino acids that do not cause loss of binding to the antigen, and which can bind to both the first and second antigens but not simultaneously; or inserting a peptide known to have binding activity to a desired antigen into the aforementioned region. Examples of peptides known to have binding activity to a desired antigen include the peptides shown in Table 1.

[0026] [Table 1]

[0027] In one aspect of the present invention, an antigen-binding molecule is provided, which includes a variable region of an antibody in which the amino acids in the heavy chain variable region have been modified so that it can bind to a first antigen and a second antigen different from the first antigen, but does not bind to the first and second antigens simultaneously. For example, by introducing the above-mentioned amino acid modification (substitution, deletion, addition, insertion, or modification, or a combination thereof) into the heavy chain variable region, a variable region of an antibody can be produced that can bind to a first antigen and a second antigen different from the first antigen, but does not bind to the first and second antigens simultaneously. The location where the amino acid modification is introduced is preferably the heavy chain variable region, and more preferably the solvent-exposed region and the loop region within the variable region. Among these, the CDR1, CDR2, CDR3, FR3 regions and the loop region are preferred. Specifically, Kabat numbering 31-35, 50-65, 71-74, and 95-102 in the H chain variable region are preferred, and Kabat numbering 31, 52a-61, 71-74, and 97-101 in the H chain variable region are more preferred. In addition, amino acids that increase antigen binding activity may be introduced during amino acid modification.

[0028] In addition to the modifications described above, the variable region of the antibody of the present invention may also include known modifications. For example, modification of the glutamine at the N-terminus of the variable region to pyroglutamic acid by pyroglutamylation is a modification well known to those skilled in the art. Therefore, if the N-terminus of the heavy chain of the antibody of the present invention is glutamine, it includes a variable region in which it is modified to pyroglutamic acid.

[0029] Furthermore, for example, amino acid modifications may be introduced to the variable region of these antibodies to improve antigen binding, pharmacokinetics, stability, and antigenicity. The variable region of the antibody of the present invention may be modified to have pH-dependent binding to the antigen, thereby enabling repeated binding to the antigen (WO / 2009 / 125825).

[0030] Furthermore, for example, amino acid modifications can be made to the variable region of these antibodies that binds to a third antigen, so that the binding activity to the antigen changes depending on the concentration of the target tissue-specific compound (WO2013 / 180200).

[0031] Furthermore, modifications to the variable region can be aimed at increasing binding activity, improving specificity, decreasing pI, conferring pH-dependent properties to antigen binding, improving binding thermal stability, improving solubility, improving stability to chemical modifications, improving heterogeneity derived from glycans, and reducing immunogenicity, as identified using in silico prediction or by in vitro T cell assays. Modifications can also be carried out to avoid T cell epitopes, or to introduce T cell epitopes that activate regulatory T cells (mAbs 3:243-247, 2011).

[0032] 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 electrochemiluminescence (ECL) (BMC Research Notes 2011, 4:281). Specifically, for example, a low-molecular-weight antibody consisting of a biotin-labeled test antigen-binding molecule with regions capable of binding to the first and second antigens, such as the Fab region, or a monovalent antibody (an antibody lacking one of the two Fab regions present in a normal antibody), is mixed with the first or second antigen labeled with a sulfo-tag (Ru complex) and added to a streptavidin-immobilized plate. At this time, the biotin-labeled test antigen-binding molecule binds to the streptavidin on the plate. By making the sulfo-tag emit light and detecting the emission signal using a Sector Imager 600, 2400 (MSD), etc., the binding of the first or second antigen to the aforementioned region of the test antigen-binding molecule can be confirmed. Furthermore, it can also be measured using methods such as ELISA, FACS (fluorescence activated cell sorting), ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), and the BIACORE method utilizing surface plasmon resonance (SPR) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0033] Specifically, for example, measurements can be performed using Biacore (GE Healthcare), an interaction analysis instrument that utilizes the surface plasmon resonance (SPR) phenomenon. This includes any Biacore model, such as Biacore T100, T200, X100, A100, 4000, 3000, 2000, 1000, and C. Any of the Biacore sensor chips, including CM7, CM5, CM4, CM3, C1, SA, NTA, L1, HPA, and Au chips, can be used. Supplementary proteins such as Protein A, Protein G, Protein L, anti-human IgG antibody, anti-human IgG-Fab, anti-human L-chain antibody, anti-human Fc antibody, antigen protein, and antigen peptide are immobilized on the sensor chip using coupling methods such as amine coupling, disulfide coupling, and aldehyde coupling to capture the antigen-binding molecule of the present invention. The first or second antigen is then passed through as an analyte, the interaction is measured, and a sensorgram is obtained. The concentration of the first or second antigen at this time can be in the range of several μM to several pM, depending on the strength of the interaction, such as the KD of the sample being measured.

[0034] Furthermore, instead of an antigen-binding molecule, it is also possible to immobilize a first or second antigen on the sensor chip and interact it with the antibody sample to be evaluated. From the dissociation constant (KD) value calculated from the interaction sensorgram, or from the degree of increase in the sensorgram before and after the antigen-binding molecule sample is applied, 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 or second antigen.

[0035] The ALPHA screen is performed using ALPHA technology, which employs two beads, a donor and an acceptor, based on the following principle: Molecules bound to the donor bead biologically interact with molecules bound to the acceptor bead, and an emission signal is detected only when the two beads are in close proximity. A photosensitiver within the donor bead, excited by a laser, converts surrounding oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and, upon reaching the nearby acceptor bead, triggers a chemiluminescent reaction within the bead, ultimately emitting light. When the molecules bound to the donor bead and the molecules bound to the acceptor bead do not interact, the singlet oxygen produced by the donor bead does not reach the acceptor bead, and therefore no chemiluminescent reaction occurs.

[0036] When one of the substances whose interaction is to be observed (ligand) is fixed onto a gold thin film on a sensor chip, and light is shone from the back of the sensor chip so as to cause total internal reflection at the interface between the gold thin film and glass, a region of reduced reflection intensity (SPR signal) is formed in a part of the reflected light. When the other substance whose interaction is to be observed (analyte) is flowed onto the surface of the sensor chip and the ligand and analyte bind, the mass of the immobilized ligand molecule increases, and the refractive index of the solvent on the surface of the sensor chip changes. This change in refractive index causes the position of the SPR signal to shift (conversely, when the binding dissociates, the signal position returns to its original position). The Biacore system plots the amount of the above shift, i.e., the change in mass on the sensor chip surface, on the vertical axis and displays the change in mass over time as measurement data (sensorgram). From the sensorgram, the amount of analyte bound to the ligand captured on the surface of the sensor chip (the change in response on the sensorgram before and after interaction with the analyte) can be determined. However, since the amount of binding also depends on the amount of ligand, when making comparisons, it is necessary to compare under conditions where the amount of ligand can be considered to be essentially the same. Furthermore, kinetics—the binding rate constant (ka) and the dissociation rate constant (kd)—can be determined from the sensorogram curve, and affinity (KD) can be determined from the ratio of these constants. Inhibition methods are also suitably used in the BIACORE method. An example of an inhibition method is described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

[0037] 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 first confirming that it has binding activity to the first antigen and the second antigen, then pre-binding either the first antigen or the second antigen to the antigen-binding molecule containing the variable region having said binding activity, and then measuring whether it has binding activity to the other antigen using the method described above. Furthermore, this can also be confirmed by measuring whether the binding of an antigen-binding molecule to either the first or second antigen, which is immobilized on an ELISA plate or sensor chip, is inhibited by adding the other antigen to the solution.

[0038] Specifically, for example, when using the ECL method, a biotin-labeled test antigen-binding molecule is prepared with a first antigen labeled with a sulfo-tag (Ru complex) and an unlabeled second antigen. If the test antigen-binding molecule can bind to both the first and second antigens, but not simultaneously, then in the absence of the unlabeled second antigen, when a mixture of the test antigen-binding molecule and the first antigen is added to a streptavidin-immobilized plate and the sulfo-tag is made to emit light, its luminescence signal is detected. However, in the presence of the second antigen, the luminescence signal decreases. By quantifying this decrease in the emission signal, the relative binding activity can be determined.

[0039] In the case of ALPHA screening, in the absence of a competing second antigen, the antigen-binding molecule and the first antigen interact, producing a signal in the 520-620 nm range. The untagged second antigen competes with the interaction between the antigen-binding molecule and the first antigen. The relative binding activity can be determined by quantifying the decrease in fluorescence resulting from this competition. Biotinylation of polypeptides using sulfo-NHS-biotin is a known method. As a method for tagging the first antigen with GST, appropriate methods may be employed, such as expressing a fusion gene (a polynucleotide encoding the first antigen and a polynucleotide encoding GST in-frame) in cells containing a vector capable of expressing it, and then purifying it using a glutathione column. The obtained signal can be suitably analyzed by fitting it to a one-site competition model utilizing nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego). In this case, the same analysis can be performed by tagging the second antigen and not tagging the first antigen. Another method utilizes fluorescence resonance energy transfer (FRET). FRET is a phenomenon in which excitation energy is directly transferred between two adjacent fluorescent molecules through electron resonance. When FRET occurs, the excitation energy of the donor (the fluorescent molecule in the excited state) is transferred to the acceptor (another fluorescent molecule located near the donor), causing the fluorescence emitted from the donor to disappear (more precisely, its fluorescence lifetime to be shortened), and instead, fluorescence is emitted from the acceptor. This phenomenon can be used to analyze whether an antibody is dual-Fab. For example, when a first antigen with a fluorescent donor and a second antigen with a fluorescent acceptor are simultaneously bound to a test antigen-binding molecule, the fluorescence of the donor disappears, and fluorescence is emitted from the acceptor, resulting in a change in fluorescence wavelength. Such antibodies are judged not to be dual-Fab. On the other hand, if, when the first antigen, the second antigen, and the test antigen-binding molecule are mixed, there is no change in the fluorescence wavelength of the fluorescent donor bound to the first antigen, then this test antigen-binding molecule can be said to be dual-Fab.

[0040] For example, a biotin-labeled antigen-binding molecule is bound to streptavidin on a donor bead, and a first antigen tagged with glutathione S-transferase (GST) is bound to an acceptor bead. In the absence of a competing second antigen, the antigen-binding molecule and the first antigen interact, producing a signal in the 520-620 nm range. The untagged second antigen competes with the interaction between the antigen-binding molecule and the first antigen. The relative binding activity can be determined by quantifying the decrease in fluorescence resulting from this competition. Biotinylation of polypeptides using sulfo-NHS-biotin is well known. As a method for tagging the first antigen with GST, appropriate methods may be employed, such as expressing a fusion gene, obtained by in-frame fusion of a polynucleotide encoding the first antigen and a polynucleotide encoding GST, in cells containing a vector capable of expressing this fusion, and purifying it using a glutathione column. The obtained signals can be suitably analyzed by fitting them to a one-site competition model that utilizes nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

[0041] Furthermore, tagging is not limited to GST; any tag is acceptable, including histidine tags, MBP, CBP, Flag tags, HA tags, V5 tags, and c-myc tags. Also, the binding of the test antigen-binding molecule to the donor beads is not limited to binding using the biotin-streptoavidin reaction. In particular, if the test antigen-binding molecule contains Fc, a method of binding the molecule via Fc-recognizing proteins such as Protein A and Protein G on the donor beads is conceivable.

[0042] Furthermore, even when the first and second antigens are not expressed on the cell membrane, such as soluble proteins, or when both are present on the same cell, simultaneous binding to both antigens is possible. However, when they are expressed on different cells, simultaneous binding is not possible, but these cases can also be measured using known methods. Specifically, even if an ECL-ELISA that detects simultaneous binding to the first and second antigens yields a positive result, if cells expressing the first antigen, cells expressing the second antigen, and the test antigen-binding molecule are mixed, and these three do not bind simultaneously, it can be shown that simultaneous binding is not possible when the antigens are expressed on different cells. For example, this can be measured using an ECL-ELISA method with cells. Cells expressing the first antigen are immobilized on a plate, the test antigen-binding molecule is attached, and then cells expressing the second antigen are added. By detecting another antigen expressed only on cells expressing the second antigen using a sulfo-tag labeled antibody, a signal is observed if the two antigens expressed on the two cells bind simultaneously, and no signal is observed if they do not bind simultaneously. Alternatively, it can be measured using the alphascreen method. When cells expressing a first antigen bound to donor beads, cells expressing a second antigen bound to acceptor beads, and a test antigen-binding molecule are mixed, a signal is observed if the two antigens expressed on the two cells bind simultaneously; if they do not bind simultaneously, no signal is observed. Alternatively, it can be measured using an interaction analysis method with Octet. First, cells expressing the first antigen, to which a peptide tag has been attached, are bound to a biosensor that recognizes the peptide tag. When interaction analysis is performed in a well containing cells expressing the second antigen and the test antigen-binding molecule, if both antigens expressed on the two cells bind simultaneously, a large wavelength shift is observed because the test antigen-binding molecule and the cell expressing the second antigen bind to the biosensor. If they do not bind simultaneously, only the test antigen-binding molecule binds to the biosensor, and a small wavelength shift is observed.

[0043] Alternatively, measurement can be performed using biological activity rather than binding activity. For example, when cells expressing a first antigen, cells expressing a second antigen, and a test antigen-binding molecule are mixed in a culture, if the two antigens expressed on the two cells bind simultaneously, they are mutually activated via the test antigen-binding molecule. This allows for the detection of changes in activation signals, such as increased phosphorylation downstream of each antigen. Alternatively, since cytokine production is induced as a result of activation, it is possible to determine whether or not the molecule binds simultaneously to the two cells by measuring the amount of cytokines produced.

[0044] In the present invention, the "Fc region" refers to a region in an antibody molecule that includes a hinge region or a part thereof, or a fragment consisting of CH2 and CH3 domains. The Fc region of an IgG class means, for example, from the 226th cysteine ​​to the C-terminus, or from the 230th proline to the C-terminus, according to EU numbering (also called EU INDEX in this specification), but is not limited to this. The Fc region can be suitably obtained by partially digesting IgG1, IgG2, IgG3, IgG4 monoclonal antibodies, etc., with a proteolytic enzyme such as pepsin, and then re-eluting the fraction adsorbed onto a protein A column or 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, for example, pepsin and papain can be given as examples.

[0045] The term "antigen-binding molecule" in this invention is not particularly limited as long as it contains the "variable region of the antibody" of this invention, and may also include peptides or proteins having a length of about 5 amino acids or more. It is not limited to peptides or proteins of biological origin, and may also include, for example, polypeptides consisting of artificially designed sequences. Furthermore, it may be a natural polypeptide, a synthetic polypeptide, a recombinant polypeptide, or any other such molecule.

[0046] A preferred example of the antigen-binding molecule of the present invention is an antigen-binding molecule that includes the Fc region of an antibody.

[0047] 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 belonging to a class of antibodies that include the same amino acid sequence as naturally occurring IgG and are 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. As for the constant region of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, multiple allotype sequences due to gene polymorphism are described in Sequences of proteins of immunological interest, NIH Publication No. 91-3242, but any of them may be used in the present invention. In particular, as for the sequence of human IgG1, the amino acid sequence at EU numbering positions 356-358 may be DEL or EEM.

[0048] Examples of antibody Fc regions include those of the IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM types. The Fc region of the antibody of the present invention can be, for example, an Fc region derived from a natural human IgG antibody. As the Fc region of the present invention, for example, an Fc region derived from the constant region of natural IgG can be used, specifically the constant region originating from natural human IgG1 (SEQ ID NO: 1), the constant region originating from natural human IgG2 (SEQ ID NO: 2), the constant region originating from natural human IgG3 (SEQ ID NO: 3), and the constant region originating from natural human IgG4 (SEQ ID NO: 4). The constant region of natural IgG also includes naturally occurring variants.

[0049] The Fc region of the present invention is preferably one in which binding activity to the Fcγ receptor is reduced. Here, the Fcγ receptor (which may be referred to as Fcγ receptor, FcγR or Fcγ receptor in this specification) is a receptor that can bind to the Fc region 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, but is not limited to, FcγRI(CD64), which includes isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII(CD32), which includes isoforms FcγRIIa (including allotypes H131 (H type) and R131 (R type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII(CD16), which includes isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any undiscovered human FcγR species or FcγR isoforms or allotypes. FcγR may be derived from any organism, but is not limited to, humans, mice, rats, rabbits, and monkeys. Mouse FcγR receptors include, but are not limited to, FcγRI(CD64), FcγRII(CD32), FcγRIII(CD16), and FcγRIII-2(CD16-2), as well as any undiscovered mouse FcγR receptors, FcγR isoforms, or allotypes. Preferred examples of such Fcγ receptors include human FcγRI(CD64), FcγRIIa(CD32), FcγRIIb(CD32), FcγRIIIa(CD16), and / or FcγRIIIb(CD16).

[0050] FcγR receptors include active receptors with an ITAM (immunoreceptor tyrosine-based activation motif) and inhibitory receptors with an ITIM (immunoreceptor tyrosine-based inhibitory motif). FcγR receptors are classified into active FcγR receptors (FcγRI, FcγRIIa R, FcγRIIa H, FcγRIIIa, FcγRIIIb) and inhibitory FcγR receptor (FcγRIIb). The polynucleotide and amino acid sequences of FcγRI are described in NM_000566.3 and NP_000557.1, respectively; the polynucleotide and amino acid sequences of FcγRIIa are described in BC020823.1 and AAH20823.1, respectively; the polynucleotide and amino acid sequences of FcγRIIb are described in BC146678.1 and AAI46679.1, respectively; the polynucleotide and amino acid sequences of FcγRIIIa are described in BC033678.1 and AAH33678.1, respectively; and the polynucleotide and amino acid sequences of FcγRIIIb are described in BC128562.1 and AAI28563.1, respectively (RefSeq registration numbers). Furthermore, FcγRIIa has 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). Similarly, FcγRIIb has 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)). Additionally, FcγRIIIa has 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)). Furthermore, FcγRIIIb has two gene polymorphisms: NA1 and NA2 (J. Clin. Invest. 85: 1287-1295 (1990)).

[0051] Whether or not the binding activity to the Fcγ receptor is reduced can be confirmed by well-known methods such as FACS, ELISA format, ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), and the BIACORE method utilizing surface plasmon resonance (SPR) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010). The ALPHA screen is performed using ALPHA technology, which employs two beads, a donor and an acceptor, based on the following principle: Molecules bound to the donor bead biologically interact with molecules bound to the acceptor bead, and a light emission signal is detected only when the two beads are in close proximity. A photosensitiver within the donor bead, excited by a laser, converts surrounding oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and, upon reaching the nearby acceptor bead, triggers a chemiluminescent reaction within the bead, ultimately emitting light. When the molecules bound to the donor bead and the molecules bound to the acceptor bead do not interact, the singlet oxygen produced by the donor bead does not reach the acceptor bead, and therefore no chemiluminescent reaction occurs.

[0052] For example, a biotin-labeled antigen-binding molecule is bound to a donor bead, and a glutathione S-transferase (GST)-tagged Fcγ receptor is bound to an acceptor bead. In the absence of competing antigen-binding molecules with mutant Fc regions, antigen-binding molecules with wild-type Fc regions interact with the Fcγ receptor, producing a signal in the 520-620 nm range. Antigen-binding molecules with untagged mutant Fc regions compete with the interaction between antigen-binding molecules with wild-type Fc regions and the Fcγ receptor. The relative binding affinity can be determined by quantifying the decrease in fluorescence resulting from this competition. Biotinylation of antigen-binding molecules such as antibodies using sulfo-NHS-biotin is well known. As a method for tagging the Fcγ receptor with GST, appropriate methods may be employed, such as expressing a fusion gene, which is an in-frame fusion of a polynucleotide encoding the Fcγ receptor and a polynucleotide encoding GST, in cells containing a vector capable of expressing this fusion, and purifying it using a glutathione column. The obtained signals can be suitably analyzed by fitting them to a one-site competition model that utilizes nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

[0053] When one of the substances whose interaction is to be observed (ligand) is fixed onto a gold thin film on a sensor chip, and light is shone from the back of the sensor chip so as to cause total internal reflection at the interface between the gold thin film and glass, a region of reduced reflection intensity (SPR signal) is formed in a part of the reflected light. When the other substance whose interaction is to be observed (analyte) is flowed onto the surface of the sensor chip and the ligand and analyte bind, the mass of the immobilized ligand molecule increases, and the refractive index of the solvent on the surface of the sensor chip changes. This change in refractive index causes the position of the SPR signal to shift (conversely, when the bond dissociates, the signal position returns to its original position). The Biacore system plots the amount of the above shift, i.e., the change in mass on the sensor chip surface, on the vertical axis and displays the change in mass over time as measurement data (sensorgram). From the sensorgram curve, kinetics: the binding rate constant (ka) and the dissociation rate constant (kd) can be determined, and affinity (KD) can be determined from the ratio of these constants. Inhibition measurement methods are also suitably used in the BIACORE method. Examples of inhibitory assay methods are described in Proc.Natl.Acad.Sci.USA (2006) 103 (11), 4005-4010.

[0054] In this specification, a decrease in binding activity to the Fcγ receptor means, for example, that, based on the analytical method described above, 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, or 1% or less, compared to the binding activity of a control antigen-binding molecule containing the Fc region. As a control antigen-binding molecule, an antigen-binding molecule having the Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be used as appropriate. The structure of the Fc region is described in SEQ ID NO: 1 (A added to the N-terminus of RefSeq registration number AAC82527.1), SEQ ID NO: 2 (A added to the N-terminus of RefSeq registration number AAB59393.1), SEQ ID NO: 3 (A added to the N-terminus of RefSeq registration number CAA27268.1), and SEQ ID NO: 4 (A added to the N-terminus of RefSeq registration number AAB59394.1). Furthermore, when using an antigen-binding molecule having a variant of the Fc region of a particular isotype of antibody as the test substance, the effect of the mutation in the variant on the binding activity to the Fcγ receptor can be verified by using an antigen-binding molecule having the Fc region of that particular isotype of antibody as a control. In this way, an antigen-binding molecule having a variant of the Fc region that has been verified to have reduced binding activity to the Fcγ receptor can be appropriately prepared.

[0055] Examples of such mutants include deletions of amino acids 231A-238S, identified 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), and C226S, C229S, E233P, L234V, L235A (Blood (2007) 109, 1185-1192). In other words, preferred antigen-binding molecules have an Fc region in which any of the following amino acids, identified according to EU numbering, among the amino acids constituting the Fc region of a particular isotype of antibody, are substituted 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, and 332. The antibody isotype from which the Fc region originates is not particularly limited, and Fc regions originating from IgG1, IgG2, IgG3, or IgG4 monoclonal antibodies can be used as appropriate, but Fc regions originating from natural human IgG1 antibodies are preferred. For example, among the amino acids constituting the Fc region of an IgG1 antibody, one of the following substitutions, identified according to EU numbering (the number represents the position of the amino acid residue identified according to EU numbering, the single amino acid symbol before the number represents the amino acid residue before substitution, and the single amino acid symbol 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 Antigen-binding molecules having an Fc region that has been modified, or an Fc region in which the amino acid sequence from position 231 to 238 is deleted, may also be used as appropriate.

[0056] Furthermore, among the amino acids constituting the Fc region of the IgG2 antibody, any of the following substitutions, identified according to EU numbering, may occur (the number represents the position of the amino acid residue identified according to EU numbering, the single amino acid symbol before the number represents the amino acid residue before substitution, and the single amino acid symbol 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 that has been modified can also be used as appropriate.

[0057] Furthermore, among the amino acids constituting the Fc region of the IgG3 antibody, any of the following substitutions can be identified according to EU numbering (the number represents the position of the amino acid residue identified according to EU numbering, the single amino acid symbol before the number represents the amino acid residue before substitution, and the single amino acid symbol after the number represents the amino acid residue before substitution); (k)F241A (l)D265A (m)V264A Antigen-binding molecules having an Fc region that has been modified can also be used as appropriate.

[0058] Furthermore, among the amino acids constituting the Fc region of the IgG4 antibody, one of the following substitutions is identified according to EU numbering (the number represents the position of the amino acid residue identified according to EU numbering, the single amino acid symbol before the number represents the amino acid residue before substitution, and the single amino acid symbol after 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 that has been modified can also be used as appropriate.

[0059] Other preferred examples include antigen-binding molecules having an Fc region in which any of the following amino acids, identified according to EU numbering, constitute the Fc region of a natural human IgG1 antibody; the 233rd, 234th, 235th, 236th, 237th, 327th, 330th, and 331st positions are substituted with the corresponding amino acids in the corresponding IgG2 or IgG4.

[0060] Other preferred examples include antigen-binding molecules having an Fc region in which one or more of the following amino acids, identified according to EU numbering, constitute the Fc region of a natural human IgG1 antibody; the 234th, 235th, and 297th positions are substituted with other amino acids. The type of amino acid present after substitution is not particularly limited, but antigen-binding molecules having an Fc region in which one or more of the 234th, 235th, and 297th positions are substituted with alanine are particularly preferred.

[0061] Other preferred examples include antigen-binding molecules having an Fc region in which the 265th position is substituted with another amino acid, among the amino acids constituting the Fc region of an IgG1 antibody, as identified according to EU numbering. The type of amino acid present after substitution is not particularly limited, but antigen-binding molecules having an Fc region in which the 265th position is substituted with alanine are particularly preferred.

[0062] Furthermore, one preferred embodiment of the "antigen-binding molecule" of the present invention is a multispecific antibody that includes the variable region of the antibody of the present invention.

[0063] To facilitate the association of multispecific antibodies, a technique can be applied that introduces electrostatic repulsion at the interface of the second constant region (CH2) or the third constant region (CH3) of the antibody H chain to suppress the association of unintended H chains (WO2006 / 106905). In a technique for suppressing unintended association of H chains by introducing electrostatic repulsion at the CH2 or CH3 interface, examples of amino acid residues that come into contact at the interface of other constant regions of the H chain include the regions corresponding to EU numbering residues 356, 439, 357, 370, 399, and 409 in the CH3 region.

[0064] More specifically, for example, in an antibody containing two types of H chain CH3 regions, one to three sets of amino acid residues selected from the following sets of amino acid residues in the first H chain CH3 region (1) to (3) can be made to have the same charge: (1) amino acid residues included in the H chain CH3 region at EU numbering positions 356 and 439; (2) amino acid residues included in the H chain CH3 region at EU numbering positions 357 and 370; (3) amino acid residues included in the H chain CH3 region at EU numbering positions 399 and 409.

[0065] Furthermore, an antibody can be formed in which one to three sets 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 which correspond 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 the opposite charge to the corresponding amino acid residues in the first H chain CH3 region.

[0066] The amino acid residues described in (1) to (3) above are in close proximity to each other when they associate. A person skilled in the art can find the site corresponding to the amino acid residues described in (1) to (3) above in a desired H chain CH3 region or H chain constant region by homology modeling using commercially available software, and can modify the amino acid residues in that site as appropriate.

[0067] In the above antibody, the "charged amino acid residue" is preferably selected from, for example, an amino acid residue belonging to either group (a) or (b) below; (a) Glutamic acid (E), aspartic acid (D), (b) Lysine (K), Arginine (R), Histidine (H).

[0068] In the above antibodies, "having the same charge" means, for example, that any of the two or more amino acid residues are amino acid residues belonging to either group (a) or (b) above. "Having opposite charges" means, for example, that if at least one of the two or more amino acid residues is an amino acid residue belonging to either group (a) or (b) above, the remaining amino acid residues are amino acid residues belonging to different groups.

[0069] In a preferred embodiment, the antibody may have a first H chain CH3 region and a second H chain CH3 region crosslinked by a disulfide bond. The amino acid residues to be modified in this invention are not limited to the amino acid residues in the variable region or constant region of the antibody as described above. Those skilled in the art can identify the amino acid residues that form an interface in polypeptide variants or heterologous polymers using homology modeling with commercially available software, and modify the amino acid residues at that site to control the association.

[0070] Furthermore, other known techniques can also be used for the association of the multispecific antibodies of the present invention. By substituting an amino acid side chain in the variable region of one H chain of the antibody with a larger side chain (knob; projection) and substituting an amino acid side chain in the opposite variable region of the other H chain with a smaller side chain (hole; void), the projection can be positioned in the void, thereby efficiently causing association between polypeptides having different amino acids and Fc regions (WO1996 / 027011, Ridgway JB et al., Protein Engineering (1996) 9, 617-621, Merchant AM et al. Nature Biotechnology (1998) 16, 677-681).

[0071] In addition, other known techniques can also be used to form the multispecific antibodies of the present invention. By using a strand-exchange engineered domain CH3, in which a portion of the CH3 of one H chain of the antibody is replaced with a sequence derived from IgA corresponding to that portion, and a sequence derived from IgA corresponding to that portion is introduced into the complementary portion of the 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). The desired multispecific antibodies can also be efficiently formed using this known technique.

[0072] Other methods for forming multispecific antibodies include antibody production techniques utilizing the association of CH1 and CL, and VH and VL of antibodies as described in WO2011 / 028952; techniques for producing bispecific antibodies using separately prepared monoclonal antibodies as described in WO2008 / 119353 and WO2011 / 131746 (Fab Arm Exchange); techniques for controlling the association between CH3 groups of antibody heavy chains as described in WO2012 / 058768 and WO2013 / 063702; techniques for producing bispecific antibodies composed of two types of light chains and one type of heavy chain as described in WO2012 / 023053; and techniques for producing bispecific antibodies using two bacterial cell lines that each express one of an antibody chain consisting of one H chain and one L chain, as described by Christoph et al. (Nature Biotechnology Vol. 31, p 753-758 (2013)). In addition to the association techniques described above, the CrossMab technique (Scaefer et al. (Proc. Natl. Acad. Sci. USA (2011) 108, 11187-11192)), known as a heterogeneous light chain association technique in which 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 are associated with a heavy chain forming a variable region that binds to a first epitope and a heavy chain forming a variable region that binds to a 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, one method can be cited in which a monoclonal antibody in which a specific amino acid present in the heavy chain CH3 region has been substituted is subjected to reducing conditions to promote the heterogeneity of the antibody and obtain the desired bispecific antibody. Preferred amino acid substitution sites in this method include, for example, the EU numbering residues 392 and 397 in the CH3 region.Furthermore, a bifunctional antibody can also be produced using an antibody in which one to three sets of amino acid residues selected from the following sets of amino acid residues in the first H chain CH3 region (1) to (3) have the same charge as the amino acid residues in the first H chain CH3 region: (1) amino acid residues in the H chain CH3 region at EU numbering positions 356 and 439; (2) amino acid residues in the H chain CH3 region at EU numbering positions 357 and 370; (3) amino acid residues in the H chain CH3 region at EU numbering positions 399 and 409. Furthermore, a bifunctional antibody can also be produced using an antibody in which one to three sets of amino acid residues selected from the above sets of amino acid residues (1) to (3) in a second H chain CH3 region different from the first H chain CH3 region have the opposite charge to the amino acid residues in the first H chain CH3 region, corresponding to the sets of amino acid residues (1) to (3) that have the same charge in the first H chain CH3 region.

[0073] Furthermore, even if it is not possible to efficiently form the desired multispecific antibody, the multispecific antibody of the present invention can also be obtained by separating and purifying the desired multispecific antibody from the produced antibody. For example, a method has been reported in which two homozygous antibodies and the desired heterozygous antibody can be purified by ion exchange chromatography by introducing amino acid substitutions into the variable regions of two types of H chains to create a difference in isoelectric points (WO2007114325). In addition, as a method for purifying heterozygous antibodies, a method has been reported in which a heterodimerized antibody consisting of a mouse IgG2a H chain that binds to protein A and a rat IgG2b H chain that does not bind to protein A is purified using protein A (WO98050431, WO95033844). Furthermore, by using H chains in which the IgG-Protein A binding sites, specifically the 435th and 436th amino acid residues in the EU numbering, are replaced with amino acids such as Tyr and His, which have different binding affinity to Protein A, the interaction between each H chain and Protein A is altered. By using a Protein A column, it is possible to efficiently purify only heterodimerized antibodies.

[0074] These techniques can be used in combination, for example, two or more. Furthermore, these techniques can be applied separately to the two H chains to be associated, as appropriate. It should be noted that the antigen-binding molecule of the present invention may also be one separately prepared, based on the modified version described above, and having the same amino acid sequence.

[0075] Amino acid sequence modifications can be carried out by various methods known in this field. 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) This can be performed by methods such as 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 US A. 82, 488-492), PCR mutation, and cassette mutation.

[0076] Furthermore, the "antigen-binding molecule" of the present invention may be an antibody fragment that contains both a heavy chain and a light chain that form the "variable region of the antibody" of the present invention within a single polypeptide chain, but lacks a constant region. Examples of such antibody fragments include diabody (Db), single-chain antibody, and sc(Fab')2.

[0077] 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.), and each polypeptide chain is linked by a linker that is short enough, for example, about 5 residues, so that the L chain variable region (VL) and H chain variable region (VH) within the same chain cannot bind to each other. Because the linker between VL and VH, which are encoded on the same polypeptide chain, is short, they cannot form a single-chain variable region fragment. Instead, they dimerize to form two antigen-binding sites.

[0078] An example of a single-chain antibody is 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 sc(Fv)2, which recognizes two different epitopes present in the same antigen, as disclosed in Journal of Immunology (1994) 152 (11), 5368-5374, is also preferred. sc(Fv)2 can be prepared by methods known to those skilled in the art. For example, it can be prepared by linking scFv with a linker such as a peptide linker.

[0079] In this specification, the antigen-binding domain configuration of sc(Fv)2 is characterized by two VHs and two VLs arranged in the order VH, VL, VH, VL ([VH]linker[VL]linker[VH]linker[VL]) starting from the N-terminus of the single-chain polypeptide. However, the order of the two VHs and two VLs is not limited to the above configuration and may be arranged in any order. For example, the following configuration can also be given. [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]

[0080] The molecular morphology of sc(Fv)2 is described in detail in WO2006 / 132352, and those skilled in the art can use these descriptions to appropriately prepare the desired sc(Fv)2 for the preparation of the antigen-binding molecules disclosed herein.

[0081] Furthermore, the antigen-binding molecule of the present invention may be conjugated with carrier polymers such as PEG or organic compounds such as anticancer agents. Additionally, a glycosylation sequence may be inserted, and the glycosylation may be suitably added to achieve a desired effect.

[0082] As the linker for binding the variable region of the antibody, any peptide linker that can be introduced by genetic engineering, or a synthetic compound linker (see, for example, Protein Engineering, 9 (3), 299-305, 1996) 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 depending on the purpose, but a 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. If sc(Fv)2 contains three peptide linkers, peptide linkers of the same length may be used, or peptide linkers of different lengths may be used.

[0083] For example, in the case of a peptide linker: Ser Gly·Ser Gly·Gly·Ser Ser·Gly·Gly Gly·Gly·Gly·Ser(Sequence ID: 5) Ser·Gly·Gly·Gly (Sequence ID: 6) Gly·Gly·Gly·Gly·Ser(Array: 7) Ser·Gly·Gly·Gly·Gly(Sequence ID: 8) Gly·Gly·Gly·Gly·Gly·Ser(Array No.: 9) Ser·Gly·Gly·Gly·Gly·Gly(Array No.: 10) Gly·Gly·Gly·Gly·Gly·Gly·Ser(Array No.: 11) Ser·Gly·Gly·Gly·Gly·Gly·Gly(Sequence ID:12) (Gly·Gly·Gly·Gly·Ser(Sequence ID:7))n (Ser·Gly·Gly·Gly·Gly(Sequence ID: 8))n Examples include [n is an integer greater than or equal to 1]. However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0084] Synthetic chemical linkers (chemical crosslinking agents) are crosslinking agents commonly used for crosslinking peptides, such as N-hydroxysuccinimide (NHS), disuccinimidylsverate (DSS), bis(sulfosuccinimidyl)sverate (BS3), dithiobis(succinimidylpropionate) (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), ethylene glycol bis(succinimidylsuccinate) (EGS), ethylene glycol bis(sulfosuccinimidylsuccinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimideoxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimideoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES), and these crosslinking agents are commercially available. When binding four antibody variable regions, typically three linkers are required, but the same linker may be used for all of them, or different linkers may be used.

[0085] F(ab')2 comprises two light chains and two heavy chains containing constant regions of the CH1 domain and a portion of the CH2 domain such that interchain disulfide bonds are formed between the two heavy chains. F(ab')2 constituting the polypeptide aggregate disclosed herein can be suitably obtained by partially digesting a full-length monoclonal antibody having a desired antigen-binding domain with a protease such as pepsin, and then removing the Fc fragment by adsorption onto a protein A column. The protease is not particularly limited as long as it can digest a full-length antibody to produce F(ab')2 restrictively by appropriately setting the reaction conditions of the enzyme, such as pH, for example, pepsin and ficin can be cited.

[0086] Furthermore, the antigen-binding molecule of the present invention may include additional modifications in addition to the amino acid modifications described above. These additional modifications can be selected from, for example, amino acid substitution, deletion, modification, or a combination thereof. For example, the antigen-binding molecule of the present invention can be further modified as desired, provided that such modification does not substantially alter the intended function of the molecule. For instance, such modifications can be made by conservative substitution of amino acid residues. Furthermore, even modifications that alter the intended function of the antigen-binding molecule of the present invention are permitted, provided that such alteration falls within the scope of the objectives of the present invention.

[0087] Modification of amino acid sequences in the present invention also includes post-translational modifications. Specific examples of post-translational modifications include the addition or deletion of glycans. For example, if the antigen-binding molecule of the present invention has a constant region of type IgG1, the 297th amino acid residue of the EU numbering may be modified with a glycan. The modified glycan structure is not limited. Generally, antibodies expressed in eukaryotic cells contain glycan modifications in their constant region. Therefore, antibodies expressed in cells such as the following are usually modified with some kind of glycan. • Mammalian antibody-producing cells • Eukaryotic cells transformed with an expression vector containing antibody-encoding DNA The eukaryotic cells shown here include yeast and animal cells. For example, CHO cells and HEK293H cells are typical animal cells used for transformation with expression vectors containing antibody-coding DNA. On the other hand, antibodies without glycosylation at that position are also included in the present invention. Antibodies whose constant region is not modified by glycosylation can be obtained by expressing the antibody-coding gene in prokaryotic cells such as E. coli.

[0088] In the present invention, additional modifications may include, more specifically, the addition of sialic acid to the sugar chain in the Fc region (MAbs. 2010 Sep-Oct;2(5):519-27.).

[0089] Furthermore, if the antigen-binding molecule of the present invention has an Fc region, amino acid substitutions may be added, for example, to improve 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), or to improve the heterogeneity and stability of the antibody ((WO / 2009 / 041613)).

[0090] Furthermore, the term "antibody" in this invention is used in its 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, and humanized antibodies, as long as they exhibit the desired biological activity.

[0091] The antibody of the present invention is not limited in terms of the type of antigen or the origin of the antibody; any antibody may be used. While not particularly limited, examples of antibody origins include human antibodies, mouse antibodies, rat antibodies, and rabbit antibodies.

[0092] Methods for producing antibodies are well known to those skilled in the art. For example, monoclonal antibodies may be produced by hybridoma (Kohler and Milstein, Nature 256:495 (1975)) or by recombinant methods (U.S. Patent No. 4,816,567). They may also be isolated from phage antibody libraries (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)).

[0093] Humanized antibodies are also called reshaped human antibodies. Specifically, known examples include humanized antibodies obtained by transplanting the CDR of an antibody from a non-human animal, such as a mouse antibody, into a human antibody. Common genetic recombination techniques for obtaining humanized antibodies are also known. Specifically, Overlap Extension PCR is a known method for transplanting the CDR of a mouse antibody into the FR of a human antibody.

[0094] A vector for humanized antibody expression can be created by inserting into an expression vector, in a way that fuses in-frame, DNA encoding an antibody variable region consisting of three CDRs and four FRs with DNA encoding a human antibody constant region. After introducing the embedded vector into a host to establish recombinant cells, the recombinant cells are cultured and the DNA encoding the humanized antibody is expressed, thereby producing the humanized antibody in the cultured cell culture (see European Patent Publication EP 239400, International Publication WO1996 / 002576).

[0095] If necessary, amino acid residues in the FR can be substituted so that the reconstituted human antibody CDR forms an appropriate antigen-binding site. For example, amino acid sequence mutations can be introduced into the FR by applying the PCR method used to transplant mouse CDRs into human FRs.

[0096] Transgenic animals possessing the entire repertoire 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.

[0097] Furthermore, a technique for obtaining human antibodies by panning using a human antibody library is also known. For example, the V region of a human antibody is expressed as a single-chain antibody (scFv) on the surface of a phage using phage display. A phage expressing an scFv that binds to an antigen can be selected. By analyzing the genes of the selected phage, the DNA sequence encoding the V region of the human antibody that binds to the antigen can be determined. After determining the DNA sequence of the scFv that binds to the antigen, an expression vector can be prepared by fusing the V region sequence in-frame with the sequence of the desired human antibody C region and then inserting it into a suitable expression vector. The human antibody can be obtained by introducing the expression vector into suitable expression cells as described above and expressing the gene encoding the human antibody. These methods are already publicly known (see International Publications WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, WO1995 / 015388).

[0098] The variable region constituting the antibody of the present invention can be a variable region that recognizes any antigen.

[0099] With the snowflakes, it's a nice place to stay It is located in the area of ​​17-IA, 4-1 BB, 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, amine, 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 B,atrial 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 TUE2), TNFSF13B (BAFF LEAD / TALL1 / THANK / TNFSF20), TNFSF14 (LIGHT WHO Ligand / LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR Ligand AITR Ligand / TL6), TNFSF1A (TNF-a Connectin / 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.

[0100] The "first antigen" and "second antigen" to which the variable region of the antibody contained in the antigen-binding molecule of the present invention binds, which can bind to two different antigens but cannot bind to these antigens simultaneously, are preferably antigens that are 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 are also expressed in normal tissues (integrin, tissue factor, VEGFR, PDGFR, EGFR, IGFR, MET chemokine receptor, heparan sulfate proteoglycan, CD44, fibronectin, DR5, TNFRSF, etc.). Preferably, the combination of the "first antigen" and "second antigen" is such that one of the first antigen or the second antigen is a molecule specifically expressed on T cells, for example, and the other antigen is a molecule expressed on the surface of T cells or other immune cells. In another embodiment, the combination of the "first antigen" and the "second antigen" is preferably such that one of the first antigen or the second antigen is a molecule specifically expressed on T cells, for example, and the other antigen is a molecule expressed on immune cells that is different from the previously selected antigen. Specifically, examples of molecules specifically expressed on T cells include CD3 and T cell receptors. CD3 is particularly preferred. The site on CD3 to which the antigen-binding molecule of the present invention binds may be any epitope present in the γ chain, δ chain, or ε chain sequence that constitutes human CD3, for example. The epitope present in the extracellular region of the ε chain of the human CD3 complex is particularly preferred. The structures of the γ, δ, or ε chains constituting CD3 are described in the following sequences: their polynucleotide sequences are in SEQ ID NOs: 83(NM_000073.2), 85(NM_000732.4), and 87(NM_000733.3), and their polypeptide sequences are in SEQ ID NOs: 84(NP_000064.1), 86(NP_000723.1), and 88(NP_000724.1) (the numbers in parentheses indicate the RefSeq registry numbers).Other antigens include Fcγ receptors, TLRs, lectins, IgA, immune checkpoint molecules, TNF superfamily molecules, TNFR superfamily molecules, and NK receptor molecules. Furthermore, the "third antigen," which is different from the "first antigen" and "second antigen" mentioned above, to which the other variable region of the antibody contained in the antigen-binding molecule of the present invention binds, is preferably an antigen specific to tumor cells. This includes antigens that are expressed in conjunction with the malignant transformation of cells, as well as abnormal sugar chains that appear on the cell surface or protein molecules when cells become cancerous. Specifically, these include, for example, ALK receptor (pleiotrophin receptor), pleiotrophin, KS 1 / 4 pancreatic cancer antigen, ovarian cancer antigen (CA125), prostatic acid phosphate, prostate-specific antigen (PSA), melanoma-related antigen p97, melanoma antigen gp75, high molecular weight melanoma antigen (HMW-MAA), prostate-specific membrane antigen, carcinogenic embryo antigen (CEA), polymorphic epithelial mucin antigen, human milk fat globule antigen, CEA, TAG-72, CO17-1A, GICA 19-9, CTA-1 and LEA, and other colorectal tumor-related antigens, as well as Burkitt lymphoma antigen-38.13, CD19, human B lymphoma antigen-CD20, CD33, melanoma-specific antigens such as ganglioside GD2, ganglioside GD3, ganglioside GM2 and ganglioside GM3, tumor-specific transplantable cell surface antigen (TSTA), T antigen, virus-induced tumor antigens such as envelope antigens of DNA oncoviruses and RNA oncoviruses, colon CEA, 5T4 carcinoembryonic trophoblast glycoprotein and bladder tumor carcinoembryonic antigen, and other carcinoembryonic α-f Etoprotein, differentiation antigens such as human lung cancer antigens L6 and L20, fibrosarcoma antigens, human leukemia T cell antigen-Gp37, neoglycoprotein, 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 in fetal red blood cells, early endoderm I antigen found in adult red blood cells, embryos before transplantation, gastric cancer I (Ma) is found in mammary epithelium, M18 and M39 are found in bone marrow cells, SSEA-1, VEP8, VEP9, Myl, and VIM-D5 are found in colorectal cancer, D156-22 and TRA-1-85 (blood group H) are found in colorectal cancer, SCP-1 is found in testicular and ovarian cancer, C14 is found in colon cancer, F3 is found in lung cancer, AH6 and Y hapten are found in gastric cancer, Ley and TL5 (blood group A) are found in embryonic cancer cells, EGF receptor is found in A431 cells, and pancreatic cancer is found The E1 series (blood group B), FC10.2 found in embryonic cancer cells, gastric cancer antigen, CO-514 (blood group Lea) found in adenocarcinoma, NS-10 found in adenocarcinoma, CO-43 (blood group Leb), G49 found in 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 embryonic cancer 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 embryos at the 4-8 cell stage, 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, paraneoplastic brain-testicular cancer antigen (carcinoma neuron antigen MA2, paraneoplastic neuron antigen), neurocarcinoma abdominal antigen 2 (NOVA2), hematological cell carcinoma antigen gene 520, tumor-associated antigen CO-029, tumor-associated antigen M Examples include AGE-C1 (cancer / testicular antigen CT7), MAGE-B1 (MAGE-XP antigen), MAGE-B2 (DAM6), MAGE-2, MAGE-4a, MAGE-4b, and MAGE-X2, cancer-testicular antigen (NY-EOS-1), YKL-40, and fragments of any of the above polypeptides or modified structures thereof (such as the modified phosphate groups and sugar chains), EpCAM, EREG, CA19-9, CA15-3, serial SSEA-1 (SLX), HER2, PSMA, CEA, CLEC12A, etc.

[0101] 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 a suitable 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 used as appropriate. Specifically, the following cells can be exemplified as animal 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. Antibodies can also be produced from E. coli (mAbs 2012 Mar-Apr; 4(2): 217-225.) and yeast (WO2000023579). Antibodies produced from E. coli do not have added sugar chains. On the other hand, antibodies produced from yeast do have added sugar chains.

[0102] The method involves expressing DNA encoding the heavy chain of an antibody, wherein 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 an antibody. 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 for a certain antigen, and introducing appropriate substitutions so that a codon encoding a specific amino acid in that region encodes another amino acid of interest.

[0103] Furthermore, it is also possible to obtain DNA encoding a heavy chain in which one or more amino acid residues in the variable region of an antibody prepared in advance using a known method for a certain antigen are substituted with other amino acids of interest, by designing DNA encoding a protein in which one or more amino acid residues in the variable region are substituted with other amino acids of interest, and then chemically synthesizing said DNA. The site of amino acid substitution and the type of substitution are not particularly limited. Preferred regions for amino acid modification include the solvent-exposed region and the loop region within the variable region. Among these, the CDR1, CDR2, CDR3, FR3 regions and the loop region are preferred. Specifically, Kabat numberings 31-35, 50-65, 71-74, 95-102 for the H chain variable region and 24-34, 50-56, 89-97 for the L chain variable region are preferred, with Kabat numberings 31, 52a-61, 71-74, 97-101 for the H chain variable region and 24-34, 51-56, 89-96 for the L chain variable region being more preferred. Furthermore, amino acid modification is not limited to substitution; it may also include deletion, addition, insertion, or modification, or a combination thereof.

[0104] Furthermore, DNA encoding a heavy chain in which one or more amino acid residues in the variable region are substituted with other amino acids of interest can be prepared by separating it into partial DNA. Examples of partial DNA combinations 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 a light chain can also be prepared by separating it into partial DNA in a similar manner.

[0105] The following methods can be used to express the above-mentioned DNA. For example, a heavy chain expression vector can be constructed by incorporating the DNA encoding the heavy chain variable region together with the DNA encoding the heavy chain constant region into an expression vector. Similarly, a light chain expression vector can be constructed by incorporating the DNA encoding the light chain variable region together with the DNA encoding the light chain constant region into an expression vector. These heavy chain and light chain genes can also be incorporated into a single vector.

[0106] When incorporating the DNA encoding the target antibody into an expression vector, it is incorporated into the expression vector so that it is expressed under the control of expression regulatory regions, such as enhancers and promoters. Next, host cells are transformed using this expression vector to express the antibody. In this process, an appropriate combination of host and expression vector can be used.

[0107] Examples of vectors include M13 vectors, pUC vectors, pBR322, pBluescript, and pCR-Script. Furthermore, for the purpose of cDNA subcloning and excision, in addition to the above vectors, other vectors such as pGEM-T, pDIRECT, and pT7 can be used.

[0108] When using a vector for the purpose of producing the antibody of the present invention, an expression vector is particularly useful. As an expression vector, for example, when the host is Escherichia coli such as JM109, DH5α, HB101, or XL1-Blue, it is essential that the vector has a promoter that can be efficiently expressed in Escherichia coli, such as the lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427, the whole of which is incorporated herein by reference), the araB promoter (Better et al., Science (1988) 240, 1041-1043, the whole of which is incorporated herein by reference), or the T7 promoter. Examples of such vectors, in addition to the above vectors, include pGEX-5X-1 (Pharmacia), "QIAexpress system" (QIAGEN), pEGFP, or pET (in this case, BL21 expressing T7 RNA polymerase is preferred as the host).

[0109] The vector may also contain a signal sequence for polypeptide secretion. For polypeptide secretion in the periplasm of *E. coli*, the pelB signal sequence (Lei, SP et al J. Bacteriol. (1987) 169, 4397, the entire sequence of which is incorporated herein by reference) may be used. The vector can be introduced into host cells using, for example, the lipofectin method, the calcium phosphate method, or the DEAE-Dextran method.

[0110] In addition to E. coli expression vectors, other vectors for producing the polypeptides of the present invention include, for example, mammalian expression vectors (e.g., pcDNA3 (Invitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18(17), p5322, the whole thereof is incorporated herein by reference), pEF, pCDM8), insect cell expression vectors (e.g., "Bac-to-BAC baculovirus expression system" (GIBCO BRL), pBacPAK8), plant expression vectors (e.g., pMH1, pMH2), animal virus expression vectors (e.g., pHSV, pMV, pAdexLcw), retrovirus expression vectors (e.g., pZIPneo), yeast expression vectors (e.g., "Pichia Expression Kit" (Invitrogen), pNV11, SP-Q01), and Bacillus subtilis expression vectors (e.g., pPL608, pKTH50).

[0111] For expression in animal cells such as CHO cells, COS cells, NIH3T3 cells, and HEK293 cells, it is essential that the vector has promoters necessary for intracellular expression, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108, the entire text is incorporated herein by reference), the MMTV-LTR promoter, the EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322, the entire text is incorporated herein by reference), the CAG promoter (Gene. (1991) 108, 193, the entire text is incorporated herein by reference), and the CMV promoter. It is even more preferable that the vector has genes for selecting transformed cells (for example, drug resistance genes that can be distinguished by drugs (neomycin, G418, etc.)). Examples of vectors having such characteristics include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13. Furthermore, in some cases, the EBNA1 protein is co-expressed to increase the copy number of the gene; in this case, a vector containing the replication origin OriP is used. (Biotechnol Bioeng. 2001 Oct 20;75(2):197-203., Biotechnol Bioeng. 2005 Sep 20;91(6):670-7.)

[0112] Furthermore, for stable gene expression and intracellular copy number amplification, one method involves introducing a vector containing the complementary DHFR gene (e.g., pCHOI) into CHO cells lacking the nucleic acid synthesis pathway, and amplifying the result with methotrexate (MTX). For transient gene expression, another method involves transforming COS cells, which have a gene expressing the SV40 T antigen on their chromosome, with a vector containing an SV40 replication origin (e.g., pcD). Replication origins can also be derived from polyomaviruses, adenoviruses, bovine papillomavirus (BPV), etc. In addition, for gene copy number amplification in the host cell line, expression vectors may include aminoglycoside transferase (APH) genes, thymidine kinase (TK) genes, Escherichia coli xanthine guanine phosphoribosyltransferase (Ecogpt) genes, dihydrofolate reductase (dhfr) genes, etc., as selection markers.

[0113] Antibodies can be recovered, for example, by culturing transformed cells and then separating them from the cells or culture medium of the molecularly transformed cells. Antibodies can be separated and purified using a combination of methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, C1q, FcRn, protein A, protein G columns, affinity chromatography, ion exchange chromatography, and gel filtration chromatography, as appropriate.

[0114] As an efficient method for producing multispecific antibodies, 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 suppressing the association of unintended H chains by introducing electrostatic repulsion (WO2006 / 106905) can be applied.

[0115] Furthermore, the present invention provides a method for producing an antigen-binding molecule comprising a variable region of an antibody capable of binding to two different first and second antigens, the variable region not binding to the first and second antigens simultaneously (first variable region), and a variable region binding to a third antigen different from the first and second antigens (second variable region), the method comprising the step of preparing an antigen-binding molecule library in which the amino acid sequences of the first variable region are diverse.

[0116] For example, a manufacturing method that includes the following steps can be cited: (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 a first antigen or a second antigen is modified, wherein at least one of the amino acids in the modified variable region comprises variable regions that are different from each other. (ii) A step of selecting from the prepared library an antigen-binding molecule that has binding activity to the first antigen and the second antigen, but contains a variable region that does not bind to the first antigen and the second antigen simultaneously. (iii) A step of culturing host cells 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, thereby expressing an antigen-binding molecule that includes a variable region of an antibody capable of binding to a first antigen and a second antigen, but to which the first and second antigens cannot bind simultaneously, and a variable region that binds to a third antigen, and (iv) A step of recovering antigen-binding molecules from the host cell culture.

[0117] In addition, in this manufacturing method, step (ii) may be one of the following optional steps: (v) A step of selecting from the prepared library an antigen-binding molecule that has binding activity to the first antigen and the second antigen, but contains a variable region that does not simultaneously bind to the first antigen and the second antigen which are expressed on different cells.

[0118] The antigen-binding molecule used in step (i) above is not particularly limited as long as it contains the variable region of the antibody, and may be an antibody fragment such as Fv, Fab, or Fab', or an antibody containing the Fc region.

[0119] For example, among the amino acids to be modified, amino acids are selected from the variable region of an antibody that binds to the first or second antigen, such that the modification does not result in the loss of binding to the antigen.

[0120] The amino acid modifications of the present invention may be used individually or in combination. When using multiple items in combination, the number of items to be combined is not particularly limited. For example, 2 to 30 items, preferably 2 to 25 items, 2 to 22 items, 2 to 20 items, 2 to 15 items, 2 to 10 items, 2 to 5 items, or 2 to 3 items. When combining multiple modifications, the amino acid modification may be applied only to the heavy chain variable region or the light chain variable region of the antibody, or it may be applied to both the heavy chain variable region and the light chain variable region as appropriate.

[0121] Preferred regions for amino acid modification include solvent-exposed regions and loop regions within the variable regions. Among these, the CDR1, CDR2, CDR3, FR3 regions and loop regions are preferred. Specifically, Kabat numberings 31-35, 50-65, 71-74, and 95-102 in the H chain variable region and Kabat numberings 24-34, 50-56, and 89-97 in the L chain variable region are preferred, with Kabat numberings 31, 52a-61, 71-74, and 97-101 in the H chain variable region and Kabat numberings 24-34, 51-56, and 89-96 in the L chain variable region being more preferred.

[0122] Furthermore, modifying amino acid residues includes randomly modifying the amino acids in the aforementioned region of the variable region of an antibody that binds to the first or second antigen, or inserting a peptide known to have binding activity to a desired antigen into the aforementioned region. In this way, by selecting a variable region from among the modified antigen-binding molecules that can bind to both the first and second antigens but cannot bind to both antigens simultaneously, 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 include the peptides shown in Table 1 above.

[0123] Whether a variable region can bind to both the first and second antigens but cannot bind to them simultaneously, and whether it can bind to both the first and second antigens simultaneously when one of them is present on a cell and the other is present alone, when both are present alone, or when both are present on the same cell, but cannot bind to them simultaneously when they are expressed on different cells, can be similarly confirmed according to the method described above.

[0124] Furthermore, the present invention provides a method for producing an antigen-binding molecule comprising a variable region of an antibody capable of binding to two different first and second antigens, but which does not bind to the first and second antigens simultaneously (a first variable region), the method comprising the step of preparing an antigen-binding molecule library in which the amino acid sequences of the first variable region are diverse.

[0125] One example of a method for producing such antigen-binding molecules is a manufacturing method that includes the following steps: (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 a first antigen or a second antigen is modified, wherein at least one of the amino acids in the modified variable region comprises variable regions that are different from each other. (ii) A step of selecting from the prepared library an antigen-binding molecule that has binding activity to the first antigen and the second antigen, but contains a variable region that does not bind to the first antigen and the second antigen simultaneously. (iii) A step of culturing host cells containing nucleic acids 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 a first antigen and a second antigen, but to which the first antigen and the second antigen cannot bind simultaneously, and (iv) A step of recovering antigen-binding molecules from the host cell culture. Furthermore, preferred regions for the above amino acid modification include the heavy chain variable region. More preferably, the solvent-exposed region and the loop region within the variable region are included. Among these, the CDR1, CDR2, CDR3, FR3 regions and the loop region are preferred. Specifically, Kabat numberings 31-35, 50-65, 71-74, 95-102 of the H chain variable region and Kabat numberings 24-34, 50-56, 89-97 of the L chain variable region are preferred, and Kabat numberings 31, 52a-61, 71-74, 97-101 of the H chain variable region and Kabat numberings 24-34, 51-56, 89-96 of the L chain variable region are more preferred.

[0126] In this manufacturing method, step (ii) above may be one of the following optional steps: (v) A step of selecting from the prepared library an antigen-binding molecule that has binding activity to the first antigen and the second antigen, but contains a variable region that does not simultaneously bind to the first antigen and the second antigen which are expressed on different cells.

[0127] The antigen-binding molecule used in step (i) above is not particularly limited as long as it contains the variable region of the antibody, and may be an antibody fragment such as Fv, Fab, or Fab', or an antibody containing the Fc region.

[0128] For example, among the amino acids to be modified, amino acids are selected from the variable region of an antibody that binds to the first or second antigen, such that the modification does not result in the loss of binding to the antigen.

[0129] The amino acid modifications of the present invention may be used individually or in combination. When using multiple items in combination, the number of items to be combined is not particularly limited. For example, 2 to 30 items, preferably 2 to 25 items, 2 to 22 items, 2 to 20 items, 2 to 15 items, 2 to 10 items, 2 to 5 items, or 2 to 3 items. When combining multiple modifications, the amino acid modification may be applied to only one of the antibody's heavy chain variable region or light chain variable region, or it may be applied to both the heavy chain variable region and the light chain variable region as appropriate.

[0130] Furthermore, modifying amino acid residues includes randomly modifying the amino acids in the aforementioned region of the variable region of an antibody that binds to the first or second antigen, or inserting a peptide known to have binding activity to a desired antigen into the aforementioned region. In this way, by selecting a variable region from among the modified antigen-binding molecules that can bind to both the first and second antigens but cannot bind to both antigens simultaneously, 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 include the peptides shown in Table 1 above.

[0131] Whether a variable region can bind to both the first and second antigens but cannot bind to them simultaneously, and whether it can bind to both the first and second antigens simultaneously when one of them is present on a cell and the other is present alone, when both are present alone, or when both are present on the same cell but cannot bind to both simultaneously when they are expressed on different cells, can be similarly confirmed according to the method described above.

[0132] Furthermore, antigen-binding molecules produced by this manufacturing method are also included in the present invention. The types and scope of amino acid modifications introduced by this method are not particularly limited.

[0133] One non-limiting aspect of the library of the present invention is a library in which 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 the library consists of antigen-binding molecules that bind to CD3 and any second antigen.

[0134] In this specification, "library" refers to multiple antigen-binding molecules or multiple fusion polypeptides containing antigen-binding molecules, or nucleic acids or polynucleotides encoding these sequences. The sequences of multiple antigen-binding molecules or multiple fusion polypeptides containing antigen-binding molecules contained in the library are not a single sequence, but rather fusion polypeptides containing antigen-binding molecules or antigen-binding molecules with different sequences from each other.

[0135] In one embodiment of the present invention, a fusion polypeptide can be prepared by combining the antigen-binding molecule of the present invention with a heterologous polypeptide. In one embodiment, the fusion polypeptide can be fused with at least a portion of viral coat proteins selected from the group consisting of, for example, pIII, pVIII, pVII, pIX, Soc, Hoc, gpD, pVI and their variants.

[0136] In one embodiment, the antigen-binding molecule of the present invention may be ScFv, Fab fragment, F(ab)2, or F(ab')2. In another embodiment, a library is provided that mainly consists of a plurality of fusion polypeptides, each having a distinct sequence from the others, in which these antigen-binding molecules are fused with a heterologous polypeptide. Specifically, a library is provided that mainly consists of a plurality of fusion polypeptides, each having a distinct sequence from the others, in which these antigen-binding molecules are fused with at least a portion of viral coat proteins selected from the group consisting of pIII, pVIII, pVII, pIX, Soc, Hoc, gpD, pVI, and their variants. The antigen-binding molecule of the present invention may further include a dimerization domain. In one embodiment, the dimerization domain may be located between the variable region of the heavy or light chain of the antibody and at least a portion of the viral coat protein. This dimerization domain may include a sequence containing at least one dimerization sequence and / or 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 take on various structures depending on whether the antibody variable region is constructed 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 constructed primarily without a viral coat protein component (for example, with an amber stop codon after the dimerization domain). When the antibody variable region is constructed primarily 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.

[0137] As used herein, the term "different in sequence from each other" in the description of a plurality of antigen-binding molecules having different sequences from each other 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 is reflected by 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 the library size can be expanded 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, which is also called the "library equivalent number", represents that there can be 10 to 10,000 individual clones having the same amino acid sequence. Thus, 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 10 6 to 10 14 molecules, preferably 10 7 to 10 12 molecules, more preferably 10 8 to 10 11 , particularly preferably 10 8 to 10 10 antigen-binding molecules that are different in sequence from each other.

[0138] 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 among the number of different independent clones of the sequences in the library. Specifically, it is preferable that there are at least 10 4 molecules of antigen-binding molecules showing such binding activity in the library. More preferably, the present invention has at least 10 5The present invention provides a library containing molecules. More preferably, the present invention provides at least 10 antigen-binding molecules exhibiting such binding activity. 6 The present invention provides a library containing molecules. Particularly preferably, the present invention provides at least 10 antigen-binding molecules exhibiting such binding activity. 7 The present invention provides a library containing molecules. Preferably, the present invention provides at least 10 antigen-binding molecules exhibiting such binding activity. 8 The present invention provides a molecular library. Alternatively, it can be preferably expressed as the proportion of antigen-binding molecules in the library that exhibit different binding activity to a first and / or second antigen, out of the total number of independent clones with different sequences. Specifically, the present invention provides a library in which antigen-binding molecules exhibiting such binding activity constitute 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 total number of independent clones with different sequences in the library. In the case of fusion polypeptides, polynucleotide molecules, or vectors, the proportion can be expressed in terms of the number of molecules or as a proportion of the total number of molecules, as described above. Similarly, in the case of viruses, the proportion can be expressed in terms of the number of individual viruses or as a proportion of the total number of individuals, as described above.

[0139] In this specification, “phage display” is a technique for presenting mutant polypeptides as proteins fused with at least a portion of the coat protein on the particle surface of a phage, such as a filamentous phage. The usefulness of phage display lies in its ability to rapidly and efficiently select sequences that bind with high affinity to a target antigen from a large library of randomized protein variants. Presentation of peptide and protein libraries on phages 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 with gene III or gene VIII of filamentous phages (Wells and Lowman (Curr. Opin. Struct. Biol. (1992) 3, 355-362) and its references). In monovalent phage display, a library of proteins or peptides is fused with gene III or a portion thereof and expressed at low levels in the presence of the wild-type gene III protein so that the phage particle presents one or zero copies of the fusion protein. Since the avidity effect is reduced compared to polyvalent phages, selection is based on endogenous ligand affinity, and phagemide vectors are used, which simplify DNA manipulation (Lowman and Wells, Methods: A Companion to Methods in Enzymology (1991) 3, 205-216).

[0140] A "phagemide" is a plasmid vector containing a bacterial origin of replication, such as ColE1, and a copy of the intergenetic region of a bacteriophage. Any known bacteriophage, such as filamentous bacteriophages and lambda bacteriophages, may be used as appropriate for the phagemide. Plasmids typically also contain a selection marker for antibiotic resistance. The DNA fragments cloned into these vectors can be grown as plasmids. When cells into which these vectors have been introduced possess all the genes necessary for the production of phage particles, the plasmid replication mode changes to rolling circle replication, producing a copy of one strand of plasmid DNA and a packaged phage particle. Phagemides can form infectious or non-infectious phage particles. The term includes phagemides containing a phage coat protein gene, or a fragment thereof, where the heterologous polypeptide is conjugated to the gene of this heterologous polypeptide as a gene fusion so that the heterologous polypeptide is presented on the surface of the phage particle.

[0141] The term "phage vector" refers to a double-stranded replicating bacteriophage that contains heterologous genes and is capable of replication. A phage vector has a phage replication origin that enables phage replication and phage particle formation. The phage is preferably a filamentous bacteriophage, such as M13, f1, fd, Pf3 phage or its derivatives, or a lambda-type phage, such as lambda, 21, phi80, phi81, 82, 424, 434, or others or their derivatives.

[0142] Oligonucleotides are short, single- or double-stranded polydeoxynucleotides chemically synthesized by known methods (e.g., solid-banding methods, phosphate triester, phosphate phosphate, or phosphoramidite chemistry using the method described in EP266032, for example, or by the method described by Froeshler et al. (Nucl. Acids. Res. (1986) 14, 5399-5407) through deoxynucleotide H-phosphonate intermediates). Other methods include polymerase chain reactions and other autoprimer 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 the entire nucleic acid sequence of a gene is known, or if a nucleic acid sequence complementary to the coding strand is available. Alternatively, if the target amino acid sequence is known, possible nucleic acid sequences can be appropriately inferred using known and preferred coding residues for each amino acid residue. Oligonucleotides can be purified using a polyacrylamide gel, a molecular sizing column, or by precipitation.

[0143] The terms "fusion protein" and "fusion polypeptide" refer to polypeptides having two parts covalently linked to each other, where each part is a polypeptide with different properties. These properties can be biological, such as in vitro or in vivo activity. They can also be single chemical or physical properties, such as binding to a target antigen or catalysis of a reaction. These two parts can be linked directly by a single peptide bond or via a peptide linker containing one or more amino acid residues. Typically, these two parts and the linker reside in the same reading frame. Preferably, the two parts of the polypeptide are obtained from heterogeneous or different polypeptides.

[0144] The term "coat protein" refers to a protein whose portion is present on the surface of a viral particle. Functionally, a coat protein is any protein that binds to a viral particle during the viral construction process in a host cell and remains bound until the virus infects another host cell. A coat protein can be a major or minor coat protein. Minor coat proteins are typically found on the outer shell of the virus, and preferably there are at least about 5 copies, more preferably at least about 7, and more preferably at least about 10 or more copies of this protein per virion. Major coat proteins can have tens, hundreds, or even thousands of copies per virion. An example of a major coat protein is the p8 protein of filamentous phages.

[0145] As an example of a non-limiting aspect of the present invention, the following six methods for creating a library are given. 1. A method for inserting a peptide (this term is used to include polypeptides and proteins) that binds to a second antigen into an antigen-binding molecule that binds to a first antigen. 2. A method for obtaining antigen-binding molecules that have binding activity to any second antigen, using the binding activity from the library to the antigen as an indicator, by creating a library in which various amino acids appear at positions that can modify (extend) the loop in the antigen-binding molecule to be longer. 3. A method for obtaining an antigen-binding molecule that has binding activity to an arbitrary second antigen, using an antigen-binding molecule whose binding activity to the first antigen is an indicator. This is done by using an antibody produced by site-directed mutagenesis from an antigen-binding molecule known to bind to a first antigen, and identifying an amino acid that maintains binding activity to the first antigen. 4.3 A method further comprising creating an antibody library in which various amino acids appear at positions that can modify (extend) the loop in the antigen-binding molecule, and obtaining an antigen-binding molecule that has binding activity to any second antigen, using the binding activity from the library to the antigen as an indicator. 5. A method of modifying the sequence in 1.2.3. or 4. to include a glycosylation sequence (e.g., NxS, NxT, where x is an amino acid other than P) and adding a glycan recognized by a glycan receptor (e.g., adding a high-mannose type glycan that is recognized by a high-mannose receptor. It is known that high-mannose type glycans can be obtained by adding kifunensin during antibody expression (MAbs. 2012 Jul-Aug;4(4):475-87)). 6. A method of adding a domain that binds to a second antigen by covalent bond by inserting or substituting Cys, Lys, or unnatural amino acids into loop sites or sites that could be modified with various amino acids in the methods of 1, 2, 3, or 4. (This method is exemplified by antibody drug conjugates, which involve covalent bonding to Cys, Lys, or unnatural amino acids (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 sites where amino acids are substituted in the antigen-binding molecule or peptides are inserted into the antigen-binding molecule are preferably the Fab or variable region of the antigen-binding molecule. Preferred regions include the solvent-exposed region and the loop region within the variable region. Among these, the CDR1, CDR2, CDR3, FR3 regions and the loop region are preferred. Specifically, Kabat numberings 31-35, 50-65, 71-74, 95-102 in the H chain variable region and Kabat numberings 24-34, 50-56, 89-97 in the L chain variable region are preferred, with Kabat numberings 31, 52a-61, 71-74, 97-101 in the H chain variable region and Kabat numberings 24-34, 51-56, 89-96 in the L chain variable region being more preferred.

[0146] As one embodiment of the method described in Method 1 above, which involves inserting a peptide that binds to a second antigen into an antigen-binding molecule that binds to a first antigen, a method of inserting G-CSF can also be mentioned, as exemplified in Angew Chem Int Ed Engl. 2013 Aug 5;52(32):8295-8. In another embodiment, the peptide to be inserted can be obtained from a library that presents peptides, but it is also possible to use all or part of a naturally occurring protein.

[0147] To identify the amino acids that maintain binding activity to the primary antigen, CD3 (in the case of human CD3, the γ, δ, or ε chains that make up human CD3), one can, for example, modify the amino acids at the site thought to be involved in antigen binding, and then produce a single-amino acid modified antibody for evaluation. For evaluating the CD3 binding of the single-amino acid modified antibody, methods known to those skilled in the art can be appropriately selected, but for example, it can be measured by ELISA, FACS (fluorescence activated cell sorting), ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), or the BIACORE method utilizing the surface plasmon resonance (SPR) phenomenon.

[0148] To identify amino acids that maintain binding activity with CD3, the results of the ratio of binding amounts of various modified compounds to the original antibody can be used. That is, if the binding amount of the original antibody is X and the binding amount of a single amino acid modified compound is Y, then the value of Z (binding amount ratio) = Y / X can be used. When Z (binding amount ratio) is 0.5 or higher, 0.6 or higher, 0.7 or higher, 0.8 or higher, 0.9 or higher, preferably 0.8 or higher, it can be considered that binding is maintained to the original antibody. Antibody libraries can be prepared so that amino acids that maintain these bindings appear.

[0149] The extracellular matrix (ECM) is one of the extracellular components and is present in various parts of the body. Therefore, antibodies that bind strongly to the ECM are known to have poor pharmacokinetics (shorter half-lives) (WO2012093704A1). For this reason, it is preferable to select amino acids that do not enhance ECM binding when selecting amino acids from antibody libraries.

[0150] To select amino acids whose ECM binding is not enhanced, for example, ECM binding can be evaluated according to the method of Reference Example 2, and the value obtained by dividing the ECM binding value (ECL response; value of ECL reaction) of each modifier by the antibody ECM binding value of MRA (H chain SEQ ID NO: 57, L chain SEQ ID NO: 58) can be used. Considering the effect of ECM binding enhancement by multiple modifications, values ​​up to 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, and 30x can be considered valid and adopted, but preferably up to 10x can be considered valid and adopted in the library. An antibody library can be prepared so that the amino acids selected in this way appear.

[0151] While not limited to this, when the peptide insertion into CDR3 is 6 amino acids, if the extended loop of CDR3 contains many amino acids with positively charged side chains, binding to the ECM is enhanced. Therefore, it is preferable that no more than 3 amino acids with positively charged side chains appear in the loop.

[0152] The library of the present invention allows for the insertion of peptides into the variable regions to enhance the diversity of the library. Preferred regions for peptide insertion include solvent-exposed regions and loop regions within the variable regions. Among these, the CDR1, CDR2, CDR3, FR3 regions and loop regions are preferred. Specifically, Kabat numbering 31-35, 50-65, 71-74, 95-102 in the H chain variable region and Kabat numbering 24-34, 50-56, 89-97 in the L chain variable region are preferred, with Kabat numbering 31, 52a-61, 71-74, 97-101 in the H chain variable region and Kabat numbering 24-34, 51-56, 89-96 in the L chain variable region being more preferred. Even more preferred is the region of Kabat numbering 99-100 in the H chain variable region. Furthermore, during amino acid modification, amino acids that increase antigen binding activity may also be introduced.

[0153] In one non-limiting aspect of the present invention, the length of the inserted peptide may be 1-3 amino acids, 4-6 amino acids, 7-9 amino acids, 10-12 amino acids, 13-15 amino acids, 15-20 amino acids, or 21-25 amino acids, but preferably 1-3 amino acids, 4-6 amino acids, or 7-9 amino acids.

[0154] To enhance library diversity, the optimal insertion sites and lengths of peptides can be investigated by creating molecules with inserted peptides and evaluating their CD3 binding. While methods known to those skilled in the art can be appropriately selected for evaluation, these can be measured using methods such as ELISA, FACS (fluorescence-activated cell sorting), ALPHA screening (Amplified Luminescent Proximity Homogeneous Assay), or the BIACORE method utilizing surface plasmon resonance (SPR).

[0155] In one non-limiting aspect of the present invention, an antibody library for obtaining antibodies that bind to CD3 and a second antigen can be designed as follows. Step 1: Select an amino acid that retains CD3 binding ability (the amount of CD3 binding should be 80% or more of that of the unmodified antibody). For example, a library can be prepared for obtaining antibodies that bind to CD3 and a second antigen, such that the amino acids selected in step 1 appear.

[0156] In one non-limiting aspect of the present invention, an antibody library for obtaining antibodies that bind to CD3 and a second antigen can be designed as follows. Step 1: Select an amino acid that retains CD3 binding ability (the amount of CD3 binding should be 80% or more of that of the unmodified antibody). Step 2: Insert amino acids between 99-100 (Kabat numbering) of the H chain CDR3. For example, by inserting amino acids into the CDR3 region in step 2 in addition to step 1, it is possible to create a library for obtaining antibodies that bind to CD3 and a second antigen, thereby enhancing the diversity of the library.

[0157] In one non-limiting aspect of the present invention, an antibody library for obtaining antibodies that bind to CD3 and a second antigen can be designed as follows. Step 1: Select an amino acid that retains CD3 binding ability (the amount of CD3 binding should be 80% or more of that of the unmodified antibody). Step 2: Select amino acids in which the ECM binding is no more than 10 times greater than in the original amino acid compared to the MRA. Step 3: Insert amino acids between 99-100 (Kabat numbering) of the H chain CDR3. For example, by adding step 2 in addition to steps 1 and 3, it is possible to select amino acids that do not enhance ECM binding from among the amino acids appearing in the library, but this method is not limited to this approach. Furthermore, even with library design that does not go through step 2, it is possible to measure and evaluate ECM binding of antigen-binding molecules obtained from the library.

[0158] In one non-limiting aspect of the present invention, when the VH region CE115HA000 (SEQ ID NO: 52) is used as the template sequence for a CD3 (CD3ε)-binding antibody, examples of amino acids used in library design include one or more amino acids from the Kabat numbering positions 11, 31, 52a, 52b, 52c, 53, 54, 56, 57, 61, 72, 78, 98, 99, 100, 100a, 100b, 100c, 100d, 100e, 100f, 100g, and 101 contained in the heavy chain variable region. The above library, in which the VH region CE115HA000 (SEQ ID NO: 52) is modified with the amino acid variant V11L / L78I, is preferred, but is not limited thereto. Furthermore, the above library, in which the VH region CE115HA000 (SEQ ID NO: 52) is modified with the amino acid variant V11L / D72A / L78I / D101Q, is preferred, but is not limited thereto.

[0159] In one non-limiting aspect of the present invention, when the VL region GLS3000 (SEQ ID NO: 53) is used as the template sequence for a CD3 (CD3ε)-binding antibody, examples of amino acids used in library design include one or more amino acids from 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, and 107 of the light chain variable region.

[0160] Designing a library in this invention includes, but is not limited to, designing a library containing multiple variants of antigen-binding molecules that include an antigen-binding domain in which an amino acid at a specific site has been modified to a desired amino acid, using known library technologies such as NNK or 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), but is not limited to this embodiment.

[0161] In the present invention, "one or more amino acids" is not particularly limited to 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 or more types of amino acids.

[0162] Regarding the presentation of fusion polypeptides, fusion polypeptides of the variable region of antigen-binding molecules can be presented in various forms on the surface of cells, viruses, or phagemide particles. These forms include single-chain Fv fragments (scFv), F(ab) fragments, and polyvalent forms of these fragments. The polyvalent 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 presentation of polyvalent forms is that they allow for the identification of typically low-affinity clones, or they have multiple antigen-binding sites that enable more efficient selection of rare clones during the selection process.

[0163] Methods for causing bacteriophages to present fusion polypeptides containing antibody fragments on their surface are known in the art and are described, for example, in WO1992001047 and herein. Related methods are also described in WO1992020791, WO1993006213, WO1993011236 and 1993019172, and these methods can be used as appropriate by those skilled in the art. Other known documents (HR Hoogenboom & G. Winter (1992) J. Mol. Biol. 227, 381-388, WO1993006213 and WO1993011236) demonstrate the identification of antibodies against various antigens presented on phage surfaces using an artificially rearranged variable region gene repertoire.

[0164] When a vector is constructed for presentation in scFv form, the vector includes nucleic acid sequences encoding the light chain variable region and heavy chain variable region of the antigen-binding molecule. Generally, the nucleic acid sequence encoding the heavy chain variable region of the antigen-binding molecule is fused to a viral coat protein component. The nucleic acid sequence encoding the light chain variable region of the antigen-binding molecule is linked to the heavy chain variable region 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 purification or detection may be fused to the 3' end of the nucleic acid sequence encoding either or both the light chain variable region or the heavy chain variable region of the antigen-binding molecule.

[0165] When a vector is constructed for presentation in the manner of F(ab), the vector includes nucleic acid sequences encoding the variable region and the constant region of the antigen-binding molecule. The nucleic acid encoding the light chain variable region is fused to the nucleic acid sequence encoding the light chain constant region. The nucleic acid sequence encoding the heavy chain variable region of the antigen-binding molecule is fused to the nucleic acid sequence encoding the heavy chain constant CH1 region. Generally, the nucleic acid sequences encoding the heavy chain variable region and the constant region are fused to the nucleic acid sequence encoding all or part of the viral coat protein. The heavy chain variable region and the constant region are preferably expressed as a fusion with at least part of the viral coat protein, while the light chain variable region and the constant region are expressed separately from the heavy chain viral coat fusion protein. The heavy and light chains bind to each other, but this binding can be covalent or non-covalent. Optionally, other sequences encoding polypeptide labels useful for purification or detection may be fused to either or both the 3' end of the nucleic acid sequence encoding the light chain constant region of the antigen-binding molecule or the 3' end of the nucleic acid sequence encoding the heavy chain constant region of the antigen-binding molecule.

[0166] Regarding the introduction of the vector into host cells, 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 and calcium phosphate precipitation. If the vector is an infectious particle such as a virus, the vector itself enters 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 by production of the phage particle by known methods.

[0167] Reproducible expression vectors can be introduced into host cells using a variety of methods. In one non-limiting embodiment, the vector can be introduced into cells using electroporation, as described in WO2000106717. The cells are cultured in a standard culture medium at 37°C for approximately 6 to 48 hours (or until the OD at 600 nm is 0.6 to 0.8), and the culture supernatant is then removed by centrifugation of the culture medium (e.g., by decantation). In 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 further centrifugation. The resulting cell pellet is resuspended in glycerin diluted to, for example, 5-20% V / V. The cell pellet is obtained by removing the supernatant from the suspension by further centrifugation. Based on the measurement of the cell concentration of the suspension obtained by resuspending the cell pellet in water or diluted glycerin, the final cell concentration is adjusted to the desired concentration using water or diluted glycerin.

[0168] For example, a preferred receptor cell is the electroporation-responsive E. coli strain SS320 (Sidhu et al. (Methods Enzymol. (2000) 328, 333-363)). E. coli strain SS320 was prepared by mating MC1061 cells with XL1-BLUE cells under conditions sufficient to transfer a fertile episome (F' plasmid) or XL1-BLUE to MC1061 cells. Deposit number 98795 has been assigned to E. 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 with ATCC, or they are commercially available (TG1, CJ236, CSH18, DHF', ER2738, JM101, JM103, JM105, JM107, JM109, JM110, KS1000, XL1-BLUE, 71-18, etc.).

[0169] Using higher DNA concentrations (approximately 10 times) in electroporation improves the transformation rate and increases the amount of DNA transformed into host cells. Using higher cell concentrations also increases efficiency (approximately 10 times). The increased amount of transferred DNA allows for the creation of larger libraries with greater diversity and a greater number of independent clones with different sequences. Transformed cells are typically selected based on their ability to grow on culture media containing antibiotics.

[0170] 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 or RNA.

[0171] Furthermore, the present invention provides a vector containing the nucleic acid of the present invention described above. The type of vector can be appropriately selected by those skilled in the art depending on the host cell into which the vector is introduced, and for example, the above-described vector can be used.

[0172] Furthermore, the present invention relates to host cells transformed with the vector of the present invention described above. The host cells can be appropriately selected by those skilled in the art, and for example, the host cells described above can be used.

[0173] Furthermore, the present invention provides a pharmaceutical composition comprising the antigen-binding molecule of the present invention and a medically acceptable carrier. The pharmaceutical composition of the present invention can be formulated by introducing a medically acceptable carrier in addition to the antigen-binding molecule of the present invention and by known methods. For example, it can be used parenterally in the form of an injectable sterile solution or suspension with water or other pharmaceutically acceptable solution. For example, it can be formulated by mixing it with a pharmacologically acceptable carrier or medium, specifically sterile water or saline solution, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc., in a unit dose form generally accepted for pharmaceutical practice. Specifically, examples of carriers include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, and inorganic salts. The amount of active ingredient in these formulations is to obtain an appropriate volume within the indicated range.

[0174] Sterile compositions for injection can be formulated in accordance with standard formulation practices using a vehicle such as distilled water for injection. Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose or other adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, which may be used in combination with appropriate solubilizers, such as alcohol, specifically ethanol, polyalcohols such as propylene glycol and polyethylene glycol, and nonionic surfactants such as polysorbate 80™ and HCO-50.

[0175] Examples of oily solutions include sesame oil and soybean oil, and may be used in combination with benzyl benzoate or benzyl alcohol as solubilizers. It may also be combined with buffers such as phosphate buffer or sodium acetate buffer, analgesics such as procaine hydrochloride, stabilizers such as benzyl alcohol or phenol, and antioxidants. The prepared injection solution is usually filled into appropriate ampoules. Administration is preferably parenteral, and specific examples include injection, nasal administration, pulmonary administration, and transdermal administration. Examples of injection formulations include intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection, which can be administered systemically or locally.

[0176] Furthermore, the method of administration can be appropriately selected depending on the patient's age and symptoms. The dosage of a pharmaceutical composition containing a polypeptide or polynucleotide encoding a polypeptide can be selected, for example, within the range of 0.0001 mg to 1000 mg per kg of body weight per dose. Alternatively, the dosage can be selected within the range of 0.001 to 100,000 mg / body per patient, but these values ​​are not necessarily the limiting factors. The dosage and method of administration will vary depending on the patient's weight, age, symptoms, etc., but those skilled in the art can select them appropriately.

[0177] Furthermore, the present invention provides a method for treating cancer, comprising 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 cancer therapeutic agent; and a process for manufacturing a cancer therapeutic agent, comprising the step of using the antigen-binding molecule of the present invention.

[0178] The correspondence between the three-letter and one-letter abbreviations for 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

[0179] It will be understood by those skilled in the art that any combination of one or more embodiments described herein is included in the present invention, insofar as it does not contradict the common technical knowledge of those skilled in the art.

[0180] All prior art documents cited herein are incorporated herein by reference.

[0181] The present invention is further illustrated by the following embodiments, but is not limited to those embodiments. [Examples]

[0182] [Example 1] Concept of a modified immunoglobulin variable (Fab) region that binds to CD3 (first antigen) and another antigen (second antigen), but does not simultaneously bind to CD3 (first antigen) and the other antigen (second antigen) on different cells. When immunoglobulins simultaneously bind to two or more molecules of activated FcγR, or when they simultaneously bind to activated FcγR with another antigen, a cross-linking reaction of activated FcγR occurs, potentially transmitting the ITAM signal of FcγR and activating immune cells. As mentioned above, one molecule of IgG antibody can only bind to one molecule of FcγR; therefore, cross-linking of two or more molecules of activated FcγR occurs only in the presence of an antigen, leading to the activation of immune cells.

[0183] Furthermore, when an IgG-type antibody binds to an antigen in its variable region (Fab), it can simultaneously bind to one molecule of FcγR in its Fc region, resulting in cross-linking between cells expressing the antigen and cells expressing FcγR. In some cases, cross-linking between the antigen and FcγR is undesirable, depending on the cells expressing the antigen. Specifically, for example, if the antigen is CD3, cross-linking of T cells with FcγR-expressing cells can lead to immune activation such as cytokine release (J. Immunol. (1999) Aug 1, 163(3), 1246-52). In such cases, it is possible to eliminate the binding activity to FcγR by introducing a modification to the Fc region, thereby preventing the cross-linking reaction between the antigen and FcγR (Advanced Drug Delivery Reviews (2006) 58, 640-656). Similarly, when the antigens of IgG antibodies are TNFR superfamily molecules such as CD40, OX40, and CD27, or TLRs such as CD3 and TLR2, 4, 8, and 9, cross-linking via FcγR can lead to systemic immune activation, making simultaneous binding to these molecules expressed on other cells undesirable.

[0184] On the other hand, conventional multispecific antibodies bind to multiple antigens simultaneously, but depending on the combination of antigens, simultaneous binding to multiple antigens may be undesirable. For example, integrin αvβ3, known as an adhesion molecule, is expressed in blood vessels surrounding many cancer cells and tumors, making it useful as a target molecule for targeting tumors (R. Haubner, PLoS Med., 2,e70(2005)). However, it is also 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 may be damaged by the potent cytotoxic activity of T cells.

[0185] Therefore, as a method to control such undesirable cross-linking reactions, a variable region (Fab) with a dual binding function (Dual Binding Fab) was conceived, in which a portion of one variable region binds to the first antigen, and another portion of the same variable region that is not involved in this binding binds to the second antigen (Figure 1). In this case, as shown in Figure 1, if two adjacent portions within a single variable region are essential for binding to their respective antigens, then when the first antigen binds, the binding of the second antigen is inhibited, and similarly, when the second antigen binds, the binding of the first antigen is inhibited. Therefore, it was thought that an improved antibody having such Dual Binding Fab properties cannot bind to both the first and second antigens simultaneously, and thus the cross-linking reaction between the first and second antigens would not occur (Figure 2). Furthermore, if the first and second antigens are not expressed on the cell membrane like soluble proteins, or if both are present on the same cell, the antibody can bind to both the first and second antigens simultaneously. However, if they are expressed on different cells, they do not bind simultaneously, and the antibody is also considered to be a Dual Binding Fab if it does not crosslink the two cells (Figure 3). On the other hand, the antigen that binds to the other variable (Fab) region (the third antigen) is thought to undergo a crosslinking reaction with the first antigen (Figure 4), and also with the second antigen (Figure 5). As the constant region of the antibody, either an Fc region that binds to FcγR can be used, or an Fc region with reduced binding activity to FcγR can be used. By utilizing the properties of such Dual Binding Fab, it becomes possible to further enhance cancer specificity in technologies that damage cancer cells expressing cancer antigens by redirecting T cells via antibodies, for example, by adding a targeting function to integrins in cancer tissue.

[0186] In other words, by improving the variable (Fab) region to create a Dual Binding Fab, if the following properties can be conferred, it will be possible to create antibodies with the effects shown in Figure 1. 1. Has binding activity to the first antigen. 2. Has binding activity to a second antigen. 3. It does not bind to the first antigen and the second antigen simultaneously. Furthermore, "not binding to the first antigen and the second antigen simultaneously" means that the protein does not cross-link the two cells expressing the first antigen and the cells expressing the second antigen, or that it does not simultaneously bind to the first antigen and the second antigen expressed on separate cells. Additionally, if the first and second antigens are not expressed on the cell membrane, like soluble proteins, or if both are present on the same cell, the protein can simultaneously bind to both the first and second antigens, but if they are expressed on different cells, simultaneous binding is not possible.

[0187] Similarly, by improving the variable (Fab) region to create a Dual Binding Fab, if the following properties can be conferred, it will be possible to create antibodies with the effects shown in Figure 6, for example. 1. Has binding activity to the first antigen on T cells. 2. It has binding activity to a second antigen on antigen-presenting cells. 3. It does not bind to the first antigen and the second antigen simultaneously.

[0188] [Example 2] Preparation of anti-human, cynomolgus monkey CD3ε antibody CE115 (2-1) Creation of hybridomas using human CD3 and cynomolgus monkey CD3-expressing cell-immunized rats 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. Taking the initial immunization as day 0, on day 0, 5 x 10¹⁶ cells were immunized with Freund's complete adjuvant (Difco). 7 Human CD3εγ-expressing Ba / F3 cells were administered intraperitoneally. On day 14, 5 x 10⁶ cells were administered with Freund's incomplete adjuvant (Difco). 7 Individual cynomolgus monkey CD3εγ-expressing Ba / F3 cells were administered intraperitoneally, followed by four weekly doses of 5 x 10⁶ cells. 7Human or cynomolgus monkey CD3εγ-expressing Ba / F3 cells were alternately administered intraperitoneally. One week after the final CD3εγ administration (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 fused using a standard method with PEG1500 (Roche Diagnostics). The fused cells, i.e., hybridomas, were cultured in RPMI1640 medium containing 10% FBS (hereinafter referred to as 10% FBS / RPMI1640).

[0189] The day after fusion, (1) the fused cells were suspended in a semi-solid medium (StemCells), and selective culture of hybridomas was performed, along with colonization of the hybridomas.

[0190] On day 9 or 10 after fusion, hybridoma colonies were picked and seeded at a rate of one colony per well in a 96-well plate containing HAT selective medium (10% FBS / RPMI1640, 2 vol% HAT 50x concentrate (Dainippon Pharmaceutical), 5 vol% BM-Condimed H1 (Roche Diagnostics)). After 3-4 days of incubation, the culture supernatant was collected from each well, and the rat IgG concentration in the culture supernatant was measured. From the culture supernatant in which rat IgG was confirmed, clones that produced antibodies specifically binding to human CD3εγ were selected by cell ELISA attached to human CD3εγ-expressing Ba / F3 cells or Ba / F3 cells that do not express human CD3εγ (Figure 7). Furthermore, cross-reactivity to monkey CD3εγ was evaluated by performing cell ELISA attached to cynomolgus monkey CD3εγ-expressing Ba / F3 cells (Figure 7).

[0191] (2-2) Production of anti-human and monkey CD3ε chimeric antibodies Total RNA was extracted from hybridoma cells using RNeasy Mini Kits (QIAGEN), and cDNA was synthesized using the SMART RACE cDNA Amplification Kit (BD Biosciences). Using the synthesized cDNA, the antibody's variable region gene was inserted into a cloning vector by PCR. The base sequence of each DNA fragment was determined using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) on an ABI PRISM 3700 DNA Sequencer (Applied Biosystems) according to the method described in the accompanying instructions. The CDR and FR 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.

[0192] A chimeric antibody H chain, formed by linking the variable region of the rat antibody H chain described above with the constant region of the human antibody IgG1 chain, and a chimeric antibody L chain gene, formed by linking the variable region of the rat antibody L chain described above with the constant region of the human antibody Kappa chain, were incorporated into an animal cell expression vector. The CE115 chimeric antibody was expressed and purified using the prepared expression vector (Reference Example 1).

[0193] (2-3) Preparation of EGFR_ERY22_CE115 Next, we created molecules using IgG as the basic skeleton for the cancer antigen (EGFR), with one of the Fabs replaced by a CD3ε-binding domain. In this case, as with the above-mentioned case, we used silent-type Fc, which has reduced binding affinity to FcgR (Fcγ receptor), as the Fc of the IgG used as the basic skeleton. For the EGFR-binding domain, we used the variable regions of Cetuximab, Cetuximab-VH (SEQ ID NO: 15) and Cetuximab-VL (SEQ ID NO: 16). As the constant region of the antibody heavy chain, G1d (IgG1 with Gly and Lys removed from the C-terminus), A5 (G1d with D356K and H435R mutations introduced), and B3 (G1d with K439E mutation introduced) were used. These were combined with Cetuximab-VH to prepare Cetuximab-VH-G1d (SEQ ID NO: 17), Cetuximab-VH-A5 (SEQ ID NO: 18), and Cetuximab-VH-B3 (SEQ ID NO: 19), respectively, according to the method of Reference Example 1. Note that if the constant region of the antibody heavy chain is named H1, the sequence corresponding to the heavy chain of an antibody with Cetuximab-VH in its variable region is indicated as Cetuximab-VH-H1, etc. Here, when indicating an amino acid modification, it is shown as D356K. The first letter (corresponding to the D in D356K) represents the letter of the original amino acid residue when expressed as a single letter, the following number (corresponding to 356 in D356K) represents the EU numbering of that modification, and the last letter (corresponding to the K in D356K) represents the letter of the modified amino acid residue when expressed as a single letter.

[0194] EGFR_ERY22_CE115 (Figure 8) was created by replacing the VH and VL domains of Fab with EGFR. Specifically, a series of expression vectors were prepared by methods known to those skilled in the art, such as PCR using primers with appropriate sequences similar to those described above, to 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.

[0195] 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

[0196] (2 - 4) Purification of EGFR_ERY₂₂_CE₁₁₅ The obtained culture supernatant was added to an Anti FLAG M2 column (Sigma), and after washing the column, elution was performed with a 0.1 mg / mL FLAG peptide (Sigma). The fraction containing the target molecule was added to a HisTrap HP column (GE Healthcare), and 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.

[0197] (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 (5,000 units of NovoSeven Heparin Injection, 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) that had been pre-injected with 15 mL of Ficoll-Paque PLUS and centrifuged. After centrifugation (2,150 rpm, 10 minutes, room temperature) of the separation tube, 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 such that the cell density became 4×10 6 / mL. The cell solution thus prepared was used in subsequent tests as a human PBMC solution.

[0198] (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 live cells was started using an xCELLigence real-time cell analyzer. The next day, the plate was removed 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 the plate was re-set in the xCELLigence real-time cell analyzer to begin measuring live cells. The reaction was carried out under 5% carbon dioxide and 37°C conditions, and the cell proliferation inhibition rate (%) was calculated from the Cell Index value 72 hours after human PBMC addition using the following formula. Note that the Cell Index value used in the calculation was the value after normalization so that the Cell Index value immediately before antibody addition was 1. Cell proliferation inhibition rate (%) = (AB) × 100 / (A - 1) A shows the average Cell Index value in wells without antibody treatment (target cells and human PBMCs only), and B shows the average Cell Index value in each well. The test was performed using a triplicate method.

[0199] When cytotoxic activity of EGFR_ERY22_CE115 using CE115 was measured with PBMCs prepared from human blood as effector cells, extremely strong activity was observed (Figure 9).

[0200] [Example 3] Production of an antibody that binds to CD3 and human integrin αvβ3 but not simultaneously. As shown in Figures 1-6, a dual-binding Fab is a molecule that binds to CD3 (the first antigen) and the target antigen (the second antigen) in its variable (Fab) region, but does not bind to both CD3 (the first antigen) and the target antigen (the second antigen) simultaneously. When introducing amino acid modifications to the Fab region of an antibody that binds to CD3 (the first antigen) in order to bind to the second antigen, the amino acid modifications are usually introduced to both the two H chains or L chains. However, if modifications are introduced to both H chains or L chains, the two Fabs of the antibody may bind to the two antigens respectively, potentially causing simultaneous binding and crosslinking between the two Fabs and both CD3 (the first antigen) and the target antigen (the second antigen). Therefore, one Fab of the antibody is designed to bind to a third antigen or not bind to anything, while the other Fab is designed as a dual-binding Fab to prevent crosslinking between CD3 (the first antigen) and the target antigen (the second antigen).

[0201] (3-1) Production of antibodies that bind to CD3 and human integrin αvβ3 but not simultaneously. Integrin αvβ3, known as an adhesion molecule, is expressed in blood vessels surrounding many cancer cells and tumors, making it a useful target molecule for tumor targeting. However, it is also known to be expressed in various normal cells (Thromb Haemost. 1998 Nov;80(5):726-34.). Therefore, it was hypothesized that if CD3 and integrin αvβ3 bind simultaneously, normal cells could be damaged by the potent cytotoxic activity of T cells. Thus, it was thought that if a molecule could be created in which CD3 and integrin αvβ3 do not bind simultaneously, anti-EGFR antibody molecules could be targeted to tumor cells expressing integrin αvβ3 without damaging normal cells. Specifically, we investigated obtaining a dual-binding Fab molecule that binds to EGFR with one variable region (Fab), binds to the first antigen, CD3, with the other variable region, and also binds to the second antigen, integrin αvβ3, while not binding to CD3 and integrin αvβ3 simultaneously.

[0202] If we can demonstrate that "a molecule in which the Fab region binds to CD3 under conditions where integrin αvβ3 is absent, and in which the Fab region binds to integrin αvβ3 under conditions where 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," then we can say that we have created a dual-binding Fab molecule that possesses the desired dual-binding Fab properties (i.e., it can bind to CD3 and a second antigen, but does not bind to CD3 and a second antigen simultaneously).

[0203] (3-2) Obtaining antibodies with a Fab region that binds to integrin αvβ3 Two methods were considered for obtaining dual-binding Fab molecules: one using a library and the other inserting peptides known to have binding activity to proteins. RGD (Arg-Gly-Asp) peptide is known to have binding activity to integrin αvβ3. Therefore, a heterodimerized antibody was prepared according to Reference Example 1, in which the RGD peptide was inserted into the loop portion of the heavy chain of CE115 (heavy chain variable region SEQ ID NO: 13, light chain variable region SEQ ID NO: 14), an antibody that binds to CD3ε, with one Fab being an EGFR-binding domain and the other a CD3-binding domain and an integrin αvβ3-binding domain. Specifically, a series of expression vectors were prepared in which polynucleotides encoding EGFR ERY22_Hk (SEQ ID NO: 20), EGFR ERY22_L (SEQ ID NO: 21), and CE115_ERY22_L (SEQ ID NO: 23), respectively, were inserted along with a polynucleotide encoding one of the following: ·CE115_2 ERY22_Hh (Sequence number: 24, Kabat numbering 52b-53 replaced with K and N respectively), ·CE115_4 ERY22_Hh (Sequence number: 25, Kabat numbering 52b-54 replaced with S and N respectively), ·CE115_9 ERY22_Hh (Sequence number: 26, RGD inserted between Kabat numbering 52a-52b), ·CE115_10 ERY22_Hh (Sequence number: 27, RGD inserted between Kabat numbering 52b-52c), ·CE115_12 ERY22_Hh (Sequence number: 28, RGD inserted between Kabat numbering 72-73), ·CE115_17 ERY22_Hh (Sequence number: 29, Kabat numbering 52b-52c replaced with K and S respectively), ·CE115_47 ERY22_Hh (Sequence number: 30, RGD inserted between Kabat numbering 98-99), ·CE115_48 ERY22_Hh (Sequence ID: 31, RGD inserted between Kabat numbering 99-100), ·CE115_49 ERY22_Hh (Sequence ID: 32, inserted into RGD between Kabat numbering 100-100a). As a control, an antibody (EH240-Kn125 / EH240-Hl076 / L73; SEQ ID NOs. 33 / 34 / 35) in which the RGD (Arg-Gly-Asp) peptide was inserted into the CH3 region of the antibody reported in J. Biotech, 155, 193-202, 2011 was prepared according to Reference Example 1. This molecule, which binds to integrin αvβ3 via the CH3 region, is thought to be able to bind to CD3 and integrin αvβ3 simultaneously.

[0204] (3-3) Confirmation of binding of integrin αvβ3 to antibody The binding of molecules with an RGD (Arg-Gly-Asp) peptide inserted into the Fab region to integrin αvβ3 was determined by electrochemiluminescence (ECL). Specifically, biotin-anti human IgG Ab (Southern biotech), diluted in a TBS solution containing 0.1% BSA, 0.1 g / L calcium chloride, and 0.1 g / L magnesium chloride (denoted as dilution (+) solution), antibody solutions prepared to 5 μg / mL or 1 μg / mL, and sulfo-tag-added integrin αvβ3 (R&D Systems) were added in 25 μL each to each well of Nunc-Immuno(tm) MicroWell(tm) 96-well round plates (Nunc). After mixing, the mixtures were incubated overnight at 4°C to form antibody-antigen complexes. A TBS solution containing 0.5% BSA, 0.1 g / L calcium chloride, and 0.1 g / L magnesium chloride (referred to as blocking(+) solution) was added to each well of a streptavidin plate (MSD) at 150 μL and incubated overnight at 4°C. After removing the blocking solution, the plates were washed three times with 250 μL of a TBS solution containing 0.1 g / L calcium chloride and 0.1 g / L magnesium chloride (referred to as TBS(+) solution). 75 μL of 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, the plates were washed three times with TBS(+) solution, 150 μL of READ buffer (MSD) was added to each well, and the emission signal of the sulfo-tag was detected using a Sector Imager 2400 (MSD).

[0205] The results are shown in Figure 11. The parent antibody EGFR ERY22_Hk / EGFR ERY22_L / CE115 ERY22_Hh / CE115_ERY22_L showed no binding activity to integrin αvβ3, whereas 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, and 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 was observed to bind to integrin αvβ3 in all cases.

[0206] (3-4) Confirmation of binding between CD3 (CD3ε) and antibody Next, the antibody that binds to integrin αvβ3 and the Fab region, prepared in the previous section, was used to determine whether it retained binding activity to CD3 by ECL. Specifically, 25 μL each of biotin-anti human IgG Ab (Southern biotech), diluted with a TBS solution containing 0.1% BSA (referred to as dilution(-) solution), antibody solutions prepared to 5 μg / mL or 1 μg / mL, and sulfo-tag-added CD3ε homodimer protein were added to each well of Nunc-Immuno(tm) MicroWell(tm) 96-well round plates (Nunc), mixed, and incubated overnight at 4°C to form antibody-antigen complexes. 150 μL each of a TBS solution containing 0.5% BSA (referred to as blocking(-) solution) was added to each well of a streptavidin plate (MSD) and incubated overnight at 4°C. After removing the blocking solution, the plates were washed three times with 250 μL of TBS (-) solution. 75 μL of antibody-antigen complex solution was added to each well, and the plates were incubated at room temperature for 2 hours to conjugate biotin-anti-human IgG Ab to the streptavidin plate. After removing the antibody-antigen complex solution, the plates were washed three times with TBS (-) solution, 150 μL of READ buffer (MSD) was added to each well, and the emission signal of the sulfo-tag was detected using a Sector Imager 2400 (MSD).

[0207] The results are shown in Figure 12. In addition to the parent 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 / Both CE115_ERY22_L were observed to bind to CD3.

[0208] (3-5) Confirmation that integrins αvβ3 and CD3 do not bind to the Fab region simultaneously using the ECL method. From the results obtained in the previous section, a molecule was obtained that has binding activity to integrin αvβ3 and also to CD3. Next, it was determined whether the Fab domain prepared in the previous section could bind simultaneously to CD3 (CD3ε) and integrin αvβ3.

[0209] When a molecule with an RGD (Arg-Gly-Asp) peptide inserted into its Fab region simultaneously binds to integrin αvβ3 and CD3, adding integrin αvβ3 and biotin-added CD3 to the antibody solution allows for detection by ECL because both antigens will bind. Specifically, 25 μL each of biotin-added human CD3ε homodimer protein diluted in dilution (+) solution, antibody solution prepared to 10 μg / mL or 5 μg / mL, and sulfo-tag-added integrin αvβ3 (R&D Systems) were added to each well of Nunc-Immuno(tm) MicroWell(tm) 96-well round plates (Nunc), mixed, and incubated overnight at 4°C to form an antibody-antigen complex. 150 μL each of blocking (+) solution was added to each well of a streptavidin plate (MSD) and incubated overnight at 4°C. After removing the blocking solution, the plates were 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 antibody-antigen conjugate solution was added to each well, and the plates were incubated at room temperature for 2 hours to conjugate biotin-anti-human IgG Ab to the streptavidin plate. After removing the antibody-antigen conjugate solution, the plates were washed three times with TBS(+) solution, 150 μL of READ buffer (MSD) was added to each well, and the emission signal of the sulfo-tag was detected using a Sector Imager 2400 (MSD).

[0210] The results are shown in FIGS. 13 and 14. 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 into the Fab region, simultaneously bound to integrin αvβ3 and CD3, and thus a strong signal was detected in the ECL measurement. On the other hand, the signals of 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 were weak (FIG. 13). In addition, almost no signal was 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 they bound to CD3.

[0211] (3-6) Consideration of why integrins αvβ3 and CD3 do not bind to the Fab region simultaneously using the ECL method. From the above results, we were able to create an antibody with the characteristics of a dual-binding Fab molecule that binds to CD3(CD3ε) and integrin αvβ3 respectively with a single Fab, but does not bind to CD3(CD3ε) and integrin αvβ3 simultaneously. In this example, by inserting an RGD peptide that binds to integrin αvβ3, a second antigen, into the variable region of an antibody that has a variable region that binds to the first antigen, CD3, we were able to confer binding activity to the second antigen and obtain a molecule that does not bind to CD3 and the second antigen simultaneously. By a similar method, a dual-binding Fab molecule with binding activity to any second antigen can be obtained by inserting a peptide that has protein-binding activity, as exemplified in WO2006036834, into the loop in the Fab. Furthermore, peptides exhibiting binding activity to proteins can be obtained by preparing a peptide library using methods known to those skilled in the art and selecting peptides with the desired activity (Pasqualini R., Nature, 1996, 380 (6572):364-6). Moreover, it is considered possible to create dual-binding Fab molecules that have binding activity to any second antigen by using a library of antigen-binding molecules in which the loop in Fab has been modified (extended) to be longer, as described in Example 5. 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 dual-binding Fab molecules that have binding activity to any first antigen and any second antigen, but cannot bind to the first and second antigens simultaneously.

[0212] 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, but do not bind to CD3 and integrin αvβ3 simultaneously. Specifically, 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 that possess dual binding Fab, and it has become clear that it is possible to create such molecules.

[0213] [Example 4] Production of an antibody that binds to CD3 and human toll-like receptor 2 (TLR2), but not simultaneously. (4-1) Production of antibodies that bind to CD3 and human TLR2 but not simultaneously. TLR2, known as a pattern recognition receptor, is mainly expressed on immune cells such as macrophages, dendritic cells, and B cells, and is useful as a target molecule for activating immune cells. Furthermore, TLR2 is known to be expressed on normal cells other than immune cells, such as epithelial cells and endothelial cells. While simultaneous binding of cancer antigens and CD3 recruits CD3-expressing T cells into the tumor environment, leading to T cell damage, it was hypothesized that simultaneous binding of cancer antigens and TLR2 could also recruit and activate immune cells expressing TLR2 into the tumor environment. Since immune cells recruited by TLR2 can engulf cancer cells damaged by T cells, process the antigen, and present it to HLA, thereby activating T cells, it may be possible to more strongly activate T cells and induce adaptive immunity. However, it was considered that simultaneous binding of CD3 and TLR2 could potentially damage immune cells and normal cells due to the potent cytotoxic activity of T cells. Therefore, it was hypothesized that if a molecule could be created that does not bind to CD3 and TLR2 simultaneously, it would be possible to recruit immune cells expressing TLR2 and normal cells without damaging them. In other words, we investigated obtaining a dual-binding Fab molecule that binds to EGFR with one variable region (Fab), binds to the first antigen, CD3, with the other variable region, and also binds to the second antigen, TLR2, while not binding to CD3 and TLR2 simultaneously.

[0214] If we can demonstrate that "a molecule in which the Fab region binds to CD3 under conditions where TLR2 is absent, and in which the Fab region binds to TLR2 under conditions where CD3 is absent, and the molecule that binds to CD3 does not bind to TLR2, or the molecule that binds to TLR2 does not bind to CD3," then we can say that we have created a dual-binding Fab molecule that possesses the desired dual-binding Fab properties (i.e., it can bind to CD3 and a second antigen, but does not bind to CD3 and a second antigen simultaneously).

[0215] (4-2) Obtaining antibodies with a Fab region that binds to TLR2 RWGYHLRDRKYKGVRSHKGVPR peptide (SEQ ID NO: 36) is known as a peptide that has binding activity to human TLR2. Therefore, a heterodimerized antibody was prepared according to Reference Example 1, in which a TRL2-binding peptide was inserted into the loop portion of the heavy chain of CE115 (heavy chain variable region SEQ ID NO: 13, light chain variable region SEQ ID NO: 14), an antibody that binds to CD3ε, with one Fab being an EGFR-binding domain and the other Fab being a CD3-binding domain and TLR2-binding domain. Specifically, a series of expression vectors were prepared in which a polynucleotide encoding one of the following was inserted along 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), respectively: ·CE115_DU21 ERY22_Hh (Sequence ID: 37, TRL2-binding peptide inserted between Kabat numbering 52b-52c), ·CE115_DU22 ERY22_Hh (Sequence ID: 38, TRL2-binding peptide inserted between Kabat numbering 52b-52c), ·CE115_DU26 ERY22_Hh (Sequence ID: 39, TRL2-binding peptide inserted between Kabat numbering 72-73), ·CE115_DU27 ERY22_Hh (Sequence ID: 40, TRL2-binding peptide inserted between Kabat numbering 72-73). As a control, an antibody with a TLR2-binding peptide attached 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 attached 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, which binds to TLR2 via the CH3 region, is thought to be able to bind to CD3 and TLR2 simultaneously.

[0216] (4-3) Confirmation of binding between TLR2 and antibody The binding of molecules with a TLR2-binding peptide inserted into the Fab region to TLR2 was determined by electrochemiluminescence (ECL). Specifically, biotin-anti human IgG Ab (Southern biotech), diluted with a TBS solution containing 0.1% BSA (referred to as dilution(-) solution), antibody solutions prepared to 5 μg / mL or 1 μg / mL, and TLR2 (abnova) with a sulfo-tag attached were added in 25 μL each to each well of Nunc-Immuno(tm) MicroWell(tm) 96-well round plates (Nunc), mixed, and incubated overnight at 4°C to form antibody-antigen complexes. 150 μL of a TBS solution containing 0.5% BSA (referred to as blocking(-) solution) was added to each well of a streptavidin plate (MSD) and incubated overnight at 4°C. After removing the blocking solution, the plates were washed three times with 250 μL of TBS(-) solution. 75 μL of antibody-antigen conjugate solution was added to each well, and the mixture was incubated at room temperature for 2 hours to conjugate biotin-anti-human IgG Ab to the streptavidin plate. After removing the antibody-antigen conjugate solution, the plates were washed three times with TBS(-) solution, and 150 μL of READ buffer (MSD) was added to each well. The emission signal of the sulfo-tag was detected using a Sector Imager 2400 (MSD).

[0217] The results are shown in Figure 15. The parent antibody EGFR ERY22_Hk / EGFR ERY22_L / CE115 ERY22_Hh / CE115_ERY22_L showed no binding activity to TLR2, whereas 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 all showed binding to TLR2.

[0218] (4-4) CD3( CD3ε ) and confirmation of antibody binding Next, the antibody prepared in the previous section, which binds to TLR2 and the Fab region, was used to determine whether it retained binding activity to CD3 (CD3ε) by ECL. Specifically, 25 μL each of biotin-anti human IgG Ab (Southern biotech), diluted with a TBS solution containing 0.1% BSA (referred to as dilution(-) solution), antibody solutions prepared to 5 μg / mL or 1 μg / mL, and sulfo-tag-added CD3ε homodimer protein were added to each well of Nunc-Immuno(tm) MicroWell(tm) 96-well round plates (Nunc), mixed, and incubated overnight at 4°C to form antibody-antigen complexes. 150 μL each of a TBS solution containing 0.5% BSA (referred to as blocking(-) solution) was added to each well of a streptavidin plate (MSD) and incubated overnight at 4°C. After removing the blocking solution, the plates were washed three times with 250 μL of TBS (-) solution. 75 μL of antibody-antigen complex solution was added to each well, and the plates were incubated at room temperature for 2 hours to conjugate biotin-anti-human IgG Ab to the streptavidin plate. After removing the antibody-antigen complex solution, the plates were washed three times with TBS (-) solution, 150 μL of READ buffer (MSD) was added to each well, and the emission signal of the sulfo-tag was detected using a Sector Imager 2400 (MSD).

[0219] The results are shown in Figure 16. In addition to the parent antibody EGFR ERY22_Hk / EGFR ERY22_L / CE115 ERY22_Hh / CE115_ERY22_L, 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 showed binding to CD3 in all cases.

[0220] (4-5) Confirmation that TLR2 and CD3 do not bind to the Fab region simultaneously using the ECL method. From the results obtained in the previous section, a molecule was obtained that has binding activity to both TLR2 and CD3. Next, it was determined whether the Fab region prepared in the previous section could bind to CD3 and TLR2 simultaneously.

[0221] When a molecule with a TLR2-binding peptide inserted into its Fab region binds simultaneously to both TLR2 and CD3, adding TLR2 and biotin-labeled CD3 to an antibody solution will cause it to bind to both antigens, allowing detection by ECL. Specifically, 25 μL each of biotin-labeled human CD3ε homodimer protein diluted in a dilution-- solution, antibody solution prepared to 10 μg / mL or 5 μg / mL, and sulfo-tag-labeled TLR2 (R&D Systems) were added to each well of Nunc-Immuno™ MicroWell™ 96-well round plates (Nunc), mixed, and incubated overnight at 4°C to form antibody-antigen complexes. 150 μL each of blocking-- solution was added to each well of a streptavidin plate (MSD) and incubated overnight at 4°C. After removing the blocking solution, the plates were 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 antibody-antigen conjugate solution was added to each well, and the plates were incubated at room temperature for 2 hours to conjugate biotin-anti-human IgG Ab to the streptavidin plate. After removing the antibody-antigen conjugate solution, the plates were washed three times with TBS(-) solution, 150 μL of READ buffer (MSD) was added to each well, and the emission signal of the sulfo-tag was detected using a Sector Imager 2400 (MSD).

[0222] The results are shown in Figure 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, which have a TLR2-binding peptide added to the CH3 region, showed a strong signal in ECL measurement due to simultaneous binding to TLR2 and CD3. On the other hand, 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 all showed almost no signal in ECL measurements. This suggests that these antibodies, when bound to CD3, do not bind to TLR2.

[0223] (4-6) Consideration of why TLR2 and CD3 do not bind to the Fab region simultaneously using the ECL method. From the above results, we were able to create an antibody with the characteristics of a dual-binding Fab molecule that binds to CD3 and TLR2 respectively with a single Fab, but does not bind to CD3 and TLR2 simultaneously. In this example, by inserting the RWGYHLRDRKYKGVRSHKGVPR peptide, which binds to the second antigen TLR2, into the Fab of an antibody having a variable region that binds to the first antigen CD3, we were able to confer binding activity to the second antigen and obtain a molecule that does not bind to CD3 and the second antigen simultaneously. By a similar method, a dual-binding Fab molecule with binding activity to any second antigen can be obtained by inserting a peptide with protein-binding activity, such as that exemplified in WO2006036834, into the loop in the Fab. In addition, peptides that exhibit protein-binding activity can be obtained by preparing a peptide library using a method known to those skilled in the art and selecting peptides with the desired activity (Pasqualini R., Nature, 1996, 380 (6572):364-6). Furthermore, by using a library of antigen-binding molecules with modified (extended) loops in the Fab, as described in Example 5, it is possible to create dual-binding Fab molecules that have binding activity to any second antigen. 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 dual-binding Fab molecules that have binding activity to any first antigen and any second antigen, but cannot bind to the first and second antigens simultaneously.

[0224] 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, but do not bind to CD3 and TLR2 simultaneously. Specifically, 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 that possess dual binding Fab, and it has become clear that it is possible to create such molecules.

[0225] [Example 5] Antibody modification for the production of antibodies that bind to CD3 and the second antigen (5-1) Examination of the insertion site and length of the peptide that can bind to the second antigen. We investigated how to obtain a dual-binding Fab molecule that binds to a cancer antigen with one variable region (Fab), binds to a first antigen (CD3) with the other variable region, and also binds to a second antigen, but does not bind to CD3 and the second antigen simultaneously. A heterodimerized antibody was prepared according to Reference Example 1, in which a GGS peptide was inserted into the loop portion of the heavy chain of CE115, an antibody that binds to CD3ε, with one Fab being an EGFR-binding domain and the other Fab being a CD3-binding domain.

[0226] In other words, between K52B and S52c in CDR2, GGS is inserted into EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE31 ERY22_Hh / CE115_ERY22_L:((Sequence ID: 20 / 21 / 43 / 23), GGSGGS peptide (Sequence ID: 90) is inserted into EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE32 ERY22_Hh / CE115_ERY22_L((Sequence ID: 20 / 21 / 44 / 23), GGSGGSGGS peptide (Sequence ID: 91) is inserted into EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE33 ERY22_Hh / CE115_ERY22_L:((Sequence IDs: 20 / 21 / 45 / 23) were created. Similarly, EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE34 ERY22_Hh / CE115_ERY22_L:((Sequence IDs: 20 / 21 / 46 / 23) with GGS inserted between D72 and D73, which are loop-shaped locations in framework 3, EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE35 ERY22_Hh / CE115_ERY22_L((Sequence IDs: 20 / 21 / 47 / 23)), and EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE36 ERY22_Hh / with GGSGGSGGS peptide (Sequence ID: 91) inserted. We created CE115_ERY22_L:((Sequence IDs: 20 / 21 / 48 / 23). Additionally, we created EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE37 ERY22_Hh / CE115_ERY22_L:((Sequence IDs: 20 / 21 / 49 / 23) with GGS inserted between A99 and Y100 in CDR3, EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE38 ERY22_Hh / CE115_ERY22_L((Sequence IDs: 20 / 21 / 50 / 23)), and EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE39 ERY22_Hh / with GGSGSGGS peptide inserted. CE115_ERY22_L:((Sequence IDs: 20 / 21 / 51 / 23) was created.

[0227] (5-2) Confirmation of binding of CE115 antibody with inserted GGS peptide to CD3ε We confirmed whether the various antibodies we prepared maintained their binding affinity to CD3ε using BiacoreT100. Biotinylated CD3ε epitope peptides were conjugated to a CM5 chip via streptavidin, and the prepared antibodies were run as analytes to analyze their binding affinity.

[0228] The results are shown in Table 2. The binding affinity of CE35, CE36, CE37, CE38, and CE39 to CD3ε was equivalent to that of the parent antibody, CE115. This indicates that it is possible to insert peptides that bind to a second antigen into these loops. Furthermore, since the binding affinity did not decrease even when GGSGGSGGS was inserted into CE36 and CE39, it was shown that the insertion of peptides of at least 9 amino acids at these sites does not affect the binding affinity to CD3ε.

[0229] [Table 2]

[0230] In other words, it was shown that by using such a peptide insertion CE115 to obtain an antibody that binds to a second antigen, it is possible to produce an antibody that can bind to CD3 and the second antigen but not simultaneously. The amino acid sequence of the peptide to be inserted or substituted here is randomly modified according to known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) or overlap extension PCR. By comparing the binding activity of each modified molecule according to the above method, a library can be prepared by determining the insertion or substitution site, the type of amino acid, and the length of the amino acid that can exhibit the desired activity even after the amino acid sequence modification.

[0231] [Example 6] Library design for obtaining antibodies that bind to CD3 and a second antigen. (6-1) Antibody libraries for obtaining antibodies that bind to CD3 and a second antigen (also known as Dual Fab Library) Six methods are given as examples of how to select CD3 (CD3ε) as the first antigen and 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 a first antigen (in addition to the peptide insertion 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 peptide or polypeptide to be bound can be obtained from the library that presents the peptide or polypeptide, but it is also possible to use all or part of a naturally occurring protein. 2. A method for obtaining Fab having binding activity to any second antigen, using the binding activity from the antibody library to the antigen as an indicator, by creating an antibody library in which various amino acids appear at positions that can be modified (extended) to lengthen the loop in Fab, as shown in Example 5. 3. A method for identifying amino acids that maintain CD3 binding activity using antibodies prepared by site-directed mutagenesis from Fab domains known to bind to CD3, and obtaining Fab domains with binding activity to any second antigen from an antibody library containing the identified amino acids, using the binding activity from the antibody library to the antigen as an indicator. In the method described in 4.3, an antibody library is prepared such that various amino acids appear at positions that can be used to modify (extend) the loop in the Fab, and a Fab having binding activity to any second antigen is obtained from the antibody library using the binding activity to the antigen as an indicator. In the method described in 5.1.2.3.4, the sequence is modified to include a glycosylation sequence (e.g., NxS, NxT, where x is an amino acid other than P), and a glycosylation sequence recognized by the glycosylation receptor is added (e.g., a high-mannose type glycosylation is added and recognized by the high-mannose receptor. It is known that high-mannose type glycosylation can be obtained by adding kifunensin during antibody expression (MAbs. 2012 Jul-Aug;4(4):475-87)). In the method described in 6.1.2.3.4, Cys, Lys, or unnatural amino acids are inserted or substituted into loop sites or sites that could be modified with various amino acids, and a domain that binds to a second antigen (a nucleic acid represented by polypeptides, glycans, or TLR agonists) is covalently added (this method, represented by Antibody drug conjugates, involves covalently attaching to Cys, Lys, or unnatural amino acids, as described in mAbs 6:1, 34-45; January / February 2014, WO2009 / 134891A2, Bioconjug Chem. 2014 Feb 19;25(2):351-61). Using the method described above, a Dual-binding Fab is obtained that binds to the first antigen and the second antigen but does not bind to each other simultaneously. This Fab can be combined with a domain that binds to any third antigen (referred to as the other variable region, as described in Example 1) by methods known to those skilled in the art, such as a common light chain, a Cross Fab, or a Fab arm exchange method.

[0232] (6-2) Production of CD3 (CD3ε)-binding antibodies with one amino acid modification using site-directed mutagenesis For the CD3 (CD3ε) binding antibody, CE115HA000 (SEQ ID NO: 52) was selected for the VH region and GLS3000 (SEQ ID NO: 53) for the VL region as template sequences. Amino acid modifications were made to the sites thought to be involved in antigen binding, according to Reference Example 1. Furthermore, the constant region of the H chain was pE22Hh (a sequence obtained by modifying the sequence from CH1 onwards of natural IgG1 with L234A, L235A, N297A, D356C, T366S, L368A, and Y407V, deleting the GK sequence at the C-terminus, and adding the DYKDDDDK sequence (SEQ ID NO: 89), SEQ ID NO: 54), and the constant region of the L chain was the Kappa chain (SEQ ID NO: 55). The modified sites are shown in Table 3. For evaluation of CD3 (CD3ε) binding activity, the one-amino acid modified antibody was obtained as a one-arm antibody (an antibody lacking one of the Fab domains of the natural IgG). Specifically, in the case of H chain modification, we used GLS3000 in which the modified H chain was linked to the constant region pE22Hh, and Kn010G3 (a modified version of the amino acid sequence from position 216 onwards of natural IgG1 with C220S, Y349C, T366W, and H435R added, SEQ ID NO: 56) and a kappa chain linked to the 3' side. In the case of L chain modification, we used a sequence in which the Kappa chain was linked to the 3' side of the modified L chain, and CE115HA000 and Kn010G3 in which pE22Hh was linked to the 3' side as the H chain. These were expressed and purified in FreeStyle293 cells (using the method of Reference Example 1).

[0233] [Table 3]

[0234] (6-3) Evaluation of CD3 binding of 1 amino acid modified antibodies The one-amino acid variants constructed and purified in (6-2) were evaluated using BiacoreT200 (GE Healthcare). An appropriate amount of CD3ε homodimer protein was immobilized on Sensor chip CM4 (GE Healthcare) by amino coupling, and then an appropriate concentration of antibody was injected as an analyte to interact with the CD3ε homodimer protein on the sensor chip. Subsequently, 10 mmol / L Glycine-HCl (pH 1.5) was injected to regenerate the sensor chip. Measurements were performed at 25 °C, and HBS-EP+ (GE Healthcare) was used as the running buffer. The measured results were used to determine the binding amount and the dissociation constant K using a single-cycle kinetics model (1:1 binding RI=0) for the sensorgram obtained from the measurement. D (M) was calculated. Biacore T200 Evaluation Software (GE Healthcare) was used to calculate each parameter.

[0235] (6-3-1) Modification of the H chain Table 4 shows the ratio of the binding amounts of various H chain variants to the original antibody, CE115HA000. Specifically, Z (the ratio of binding amounts) = Y / X, where X is the binding amount of the antibody containing CE115HA000 and Y is the binding amount of the H chain 1-amino acid variant. In this case, as shown in Figure 18, if Z is less than 0.8, the sensor gram indicates a very low binding amount, and the dissociation constant K is correctly identified. D It was suggested that (M) could not be calculated. Next, the dissociation constant K of various H chain variants for CE115HA000. D Table 5 shows the ratio of (M) (=KD value of CE115HA000 / KD value of the modified version). As shown in Table 4, if Z is 0.8 or higher, it is considered that the antibody maintains binding to the original antibody CE115HA000. Therefore, an antibody library designed to produce these amino acids can be a Dual Fab Library.

[0236] [Table 4]

[0237] [Table 5]

[0238] (6-3-2) Modification of the L chain Table 6 shows the ratio of the binding amount of various L-chain variants to the original antibody, GLS3000. Specifically, Z (ratio of binding amounts) = Y / X, where X is the binding amount of the antibody containing GLS3000 and Y is the binding amount of the L-chain 1-amino acid variant. In this case, as shown in Figure 18, if Z is less than 0.8, the sensor gram indicates a very low binding amount, and the dissociation constant K is correctly identified. D It was suggested that (M) could not be calculated. Next, the dissociation constant K of various L-chain variants for GLS3000. D The ratio of (M) is shown in Table 7. As shown in Table 6, if Z is 0.8 or higher, it is considered that the antibody maintains binding to the original antibody, GLS3000. Therefore, an antibody library designed to produce these amino acids can be a Dual Fab Library.

[0239] [Table 6]

[0240] [Table 7]

[0241] (6-4) Evaluation of ECM (Extracellular Matrix) Binding of Single-Amino Acid Modified Antibodies The extracellular matrix (ECM) is one of the extracellular components and is present in various parts of the body. Therefore, antibodies that bind strongly to the ECM are known to have poor pharmacokinetics (shorter half-lives) (WO2012093704A1). For this reason, it is preferable to select amino acids that do not enhance ECM binding when selecting amino acids from antibody libraries.

[0242] Antibodies were obtained from each H-chain or L-chain variant using the method shown in (6-2). Next, ECM binding was evaluated according to the method of Reference Example 2. The ECM binding value (ECL response; value of ECL reaction) of each variant was divided by the antibody ECM binding value of MRA (H-chain SEQ ID NO: 57, L-chain SEQ ID NO: 58) in the same plate or on the same day, and the results 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 variants. Of the values ​​shown in Table 8 (H chain) and Table 9 (L chain), considering the effect of ECM binding enhancement through multiple modifications, values ​​up to 10 times were considered valid and adopted for the Dual Fab Library.

[0243] [Table 8]

[0244] [Table 9]

[0245] (6-5) Examination of peptide insertion sites and lengths to enhance library diversity In Example 5, it was demonstrated that peptides could be inserted at each location using GGS sequences without losing binding to CD3 (CD3ε). It was hypothesized that if loop extension were possible in a Dual Fab Library, it would result in a library containing a wider variety of molecules (also described as having greater diversity), enabling the acquisition of Fab domains that bind to diverse secondary antigens. Therefore, since a decrease in binding activity was expected with peptide insertion, a molecule was created by modifying the CE115HA000 sequence with V11L / D72A / L78I / D101Q to enhance CD3ε binding activity, ligating pE22Hh, and then inserting a GGS linker in the same manner as in Example 5. CD3 binding was then evaluated. The GGS sequence was inserted between Kabat numbering 99-100. The antibody molecule was expressed as a one-arm antibody. Specifically, a sequence was adopted in which the aforementioned H chain containing the GGS linker was linked with Kn010G3 (SEQ ID NO: 56) and the L chain consisted of GLS3000 (SEQ ID NO: 53) and a Kappa sequence (SEQ ID NO: 55), and expression purification was performed according to Reference Example 1.

[0246] (6-6) Confirmation of binding of CE115 antibody with inserted GGS peptide to CD3 The binding of the modified antibody with inserted GGS peptide to CD3ε was performed using Biacore as described in Example 6. As a result, it was revealed that insertion of the GGS linker into the loop region is possible, as shown in Table 10. In particular, it was possible to insert the GGS linker into the H 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, we investigated using a GGS linker, but it is thought that antibody libraries containing various amino acids instead of GGS could also be used.

[0247] [Table 10]

[0248] (6-7) Investigation of library insertion into H chain CDR3 using NNS bases In (6-6), it was thought that 3, 6, and 9 amino acids could be inserted using the GGS linker, and that by creating a library with 3, 6, and 9 amino acids inserted and using an antibody acquisition method such as the standard phase display method, an antibody that binds to a second antigen could be obtained. Therefore, when 6 amino acids were inserted into CDR3, we investigated whether binding to CD3 could be maintained even if various amino acids appeared at the insertion site 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 were inserted between 99-100 (Kabat numbering) in the CDR3 of the CE115HA340 sequence (SEQ ID NO: 59), which has higher CD3ε binding activity than CE115HA000, with higher CD3ε binding activity. The antibody molecule was expressed as a one-arm antibody. Specifically, a sequence was adopted in which the aforementioned modified H chain, Kn010G3 (SEQ ID NO: 56), and GLS3000 (SEQ ID NO: 53) and a Kappa sequence (SEQ ID NO: 55) were linked as the L chain, and expression purification was performed according to Reference Example 1. The obtained modified antibody was evaluated for binding using the method described in (6-3). The results are shown in Table 11. It was found that binding to CD3 (CD3ε) was maintained even when various amino acids appeared in the extended amino acid site. Furthermore, the results of evaluating whether nonspecific binding was enhanced using the method shown in Reference Example 2 are shown in Table 12. As a result, it was found that binding to the ECM was enhanced when many amino acids with positive charges in their side chains were included in the extended loop of CDR3, so it was desirable that no more than three amino acids with positive charges in their side chains appeared in the loop.

[0249] [Table 11]

[0250] [Table 12]

[0251] (6-7) Design and construction of a Dual Fab Library Based on 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 an amino acid that retains CD3 (CD3ε) binding ability (the amount of CD3 binding should be 80% or more of that of CE115HA000). Step 2: Select amino acids in which the ECM binding is no more than 10 times greater than in the original amino acid compared to the MRA. Step 3: Insert 6 amino acids between 99-100 (Kabat numbering) of the H chain CDR3. Furthermore, even with only Step 1, the antigen-binding sites of Fab are diversified, making it possible to create a library for identifying antigen-binding molecules that bind to a second antigen. Also, even with only Steps 1 and 3, the antigen-binding sites of Fab are diversified, making it possible to create a library for identifying antigen-binding molecules that bind to a second antigen. Even with library design that does not go through Step 2, ECM binding can be measured and evaluated for the obtained molecules.

[0252] From the above, the H chain of the Dual Fab Library was diversified as shown in Table 13, using a sequence with the V11L / L78I mutation added to the FR (framework) of CE115HA000 as the CDR, and the L chain was diversified as shown in Table 14, using the CDR of GLS3000. These antibody library fragments can be synthesized using DNA synthesis methods known to those skilled in the art. As Dual Fab libraries, it is possible to create libraries in the following ways: (1) a library in which the H chain is diversified as shown in Table 13 and the L chain is fixed to the original sequence GLS3000 or the CD3ε-enhanced L chain described in Example 6; (2) a library in which the H chain is fixed to the original sequence (CE115HA000) or the CD3ε-enhanced H chain described in Example 6 and the L chain is diversified as shown in Table 14; and (3) a library in which the H chain is diversified as shown in Table 13 and the L chain is diversified as shown in Table 14. The H chain was derived from the CE115HA000 FR (framework) sequence with the V11L / L78I mutation, and the resulting diversified library sequence, as shown in Table 13, was commissioned to a DNA2.0 DNA synthesis company to obtain antibody library fragments (DNA fragments). The obtained antibody library fragments were amplified by PCR and inserted into phage display phagemides. GLS3000 was selected as the L chain for this process. The constructed phage display phagemides were then introduced into E. coli by electroporation to create E. coli cells containing the antibody library fragments.

[0253] [Table 13]

[0254] [Table 14]

[0255] [Example 7] Obtaining Fab domains that bind to CD3 and a second antigen (IL6R) from a Dual Fab Library (7-1) Acquisition of a Fab domain that binds to human IL6R Fab domains (antibody fragments) that bind to human IL6R were identified from the Dual Fab library designed and constructed in Example 6. Using biotin-labeled human IL6R as the antigen, antibody fragments that bind to human IL6R were enriched. Phage production was induced from E. coli containing the constructed phage display phagemide. A phage library solution was obtained by adding 2.5 M NaCl / 10% PEG to the culture medium of the phage-producing E. coli, precipitating the phage population, and then diluting the precipitate with TBS. Next, BSA was added to the phage library solution to a final concentration of 4% BSA. For panning, the common method of panning using antigens immobilized on magnetic beads was referenced (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). NeutrAvidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or Streptavidin-coated beads (Dynabeads M-280 Streptavidin) were used as magnetic beads.

[0256] 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 antigen-phage complex was bound to the magnetic beads at room temperature for 15 minutes. The beads were washed three times with TBST (TBS containing 0.1% Tween20, manufactured by TaKaRa), and then washed two more times with 1 mL of TBS. Subsequently, 0.5 mL of 1 mg / mL of trypsin was added to the beads, and after being suspended at room temperature for 15 minutes, the beads were immediately separated using a magnetic stand, and the phage solution was recovered. The recovered phage solution was added to 10 mL of E. coli strain ER2738 in the logarithmic growth phase (OD600 0.4-0.5). The E. coli was slowly cultured with agitation at 37°C for 1 hour to infect the E. coli with the phages. Infected E. coli were seeded onto a 225 mm x 225 mm plate. Next, a phage library was prepared by recovering phages from the culture medium of the seeded E. coli. This cycle, called panning, was repeated multiple times. For the second and subsequent panning cycles, 40 pmol of biotin-labeled antigen was used. In the fourth panning cycle, phage enrichment was performed using CD3 binding as an indicator. Specifically, 250 pmol of biotin-labeled CD3ε peptide antigen (amino acid sequence number: 60) was added to the prepared phage library, and the phage library was contacted with the antigen at room temperature for 60 minutes. Magnetic beads blocked with BSA were added, and the antigen-phage complex was bound to the magnetic beads at room temperature for 15 minutes. The beads were washed with 1 mL of 0.1% Tween20-containing TBS and TBS. Beads to which 0.5 mL of 1 mg / mL trypsin was added were suspended at room temperature for 15 minutes, after which the beads were immediately separated using a magnetic stand and the phage solution was recovered. The phages recovered from the trypsin-treated phage solution were added to 10 mL of E. coli strain ER2738 in the logarithmic growth phase (OD600 0.4-0.7). The E. coli was slowly cultured with agitation at 37°C for 1 hour to infect the E. coli with the phages.Infected E. coli were seeded onto 225 mm x 225 mm plates. Next, a phage library was collected by recovering phages from the culture medium of the seeded E. coli. Furthermore, to prevent multiple phages from infecting a single E. coli bacterium, a phage library prepared from E. coli infected with phages recovered during the fifth panning was used to re-infect E. coli with a phage solution diluted 100,000 times, and single colonies were obtained by infecting E. coli with this solution.

[0257] (7-2) Binding of the Fab domain presented by the phage to CD3 or IL6R (phage ELISA method) From the single colonies of E. coli obtained by the method described above, phage-containing culture supernatant was collected according to the standard method (Methods Mol. Biol. (2002) 178, 133-145). The phage-containing culture supernatant, to which BSA was added to a final concentration of 4% BSA, was subjected to ELISA using the following procedure. A StreptaWell 96 microtiter plate (Roche) was coated with 100 μL of PBS containing biotin-labeled antigen (biotinylated CD3ε peptide or biotinylated human IL6R) at 4°C overnight or at room temperature for 1 hour. After removing the antigen by washing each well of the plate with PBST, the wells were blocked with 250 μL of 4% BSA-TBS for at least 1 hour. The prepared culture supernatant was added to each well from which the 4% BSA-TBS had been removed, and the plate was allowed to stand at room temperature for 1 hour to allow antibodies that present phages to bind to the antigens present in each well. After washing each well with TBST, HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech), diluted with TBS to a final concentration of 4% BSA, was added and incubated for 1 hour. After washing with TBST, the color reaction of the solution in each well to which TMB single solution (ZYMED) was added was stopped by the addition of sulfuric acid, and the color development was measured by absorbance at 450 nm. The results are shown in Figure 19. Clones #50 and #62 were shown to bind to CD3ε and human IL6R. That is, by using the Dual Fab Library, it was possible to select clones that show binding to the second antigen (human IL6R in Example 7). Furthermore, by increasing the number of evaluations and selecting clones that show binding, it is possible to IgGify them (linking the VH and VL sequences of the clone to the human H chain or L chain constant region, respectively) and evaluate their binding to CD3ε and the second antigen (human IL6R). Furthermore, whether CD3ε and the second antigen (human IL6R) bind simultaneously can be investigated using the methods described in Examples 3 and 4, as well as the competitive method. The competitive method demonstrates that simultaneous binding does not occur, for example, by reducing the binding to CD3ε when the second antigen is present compared to when the antibody is alone.

[0258] [Example 8] Obtaining Fab domains that bind to CD3 and a second antigen (human IgA) from a Dual Fab Library (8-1) Obtaining a Fab domain that binds to human IgA IgA is an isotype of antibody that is abundant in the body and is known to be a molecule involved in biological defense in the intestines and mucous membranes, and is known to bind to FcαR (Fc alpha receptor) (J. Pathol. 208: 270 -282, 2006). Fab domains (antibody fragments) that bind to human IgA were identified from the Dual Fab library designed and constructed in Example 6. Using biotin-labeled human IgA (described in Reference Example 3) as the antigen, antibody fragments that bind to human IgA were enriched. Phage production was induced from E. coli containing the constructed phage display phagemide. A phage library solution was obtained by adding 2.5 M NaCl / 10% PEG to the culture medium of the phage-producing E. coli, precipitating the phage population, and then diluting the precipitate with TBS. Next, BSA was added to the phage library solution to a final concentration of 4% BSA. For panning, the common method of panning using antigens immobilized on magnetic beads was referenced (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). NeutrAvidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or Streptavidin-coated beads (Dynabeads M-280 Streptavidin) were used as magnetic beads. 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 antigen-phage complex was bound to the magnetic beads at room temperature for 15 minutes. The beads were washed three times with TBST (TBS containing 0.1% Tween20, manufactured by TaKaRa), and then washed two more times with 1 mL of TBS. Subsequently, 0.5 mL of 1 mg / mL of trypsin was added to the beads, and after being suspended at room temperature for 15 minutes, the beads were immediately separated using a magnetic stand, and the phage solution was recovered. The recovered phage solution was added to 10 mL of E. coli strain ER2738 in the logarithmic growth phase (OD600 0.4-0.5). The E. coli was slowly cultured with agitation at 37°C for 1 hour to infect the E. coli with the phages. Infected E. coli were seeded onto 225 mm x 225 mm plates. Next, a phage library was prepared by recovering phages from the culture medium of the seeded E. coli. This cycle, called panning, was repeated four times. For the second and subsequent panning cycles, the human IgA concentration was set to 40 pmol.

[0259] (8-2) Binding of the Fab domain presented by the phage to CD3 or human IgA From the single colonies of E. coli obtained by the method described above, the phage-containing culture supernatant was collected according to the standard method (Methods Mol. Biol. (2002) 178, 133-145). The phage-containing culture supernatant, to which BSA was added to a final concentration of 4% BSA, was subjected to ELISA according to the following procedure. A StreptaWell 96 microtiter plate (Roche) was coated with 100 μL of PBS containing biotin-labeled antigen (biotin-labeled CD3ε peptide or biotin-labeled human IgA, Reference Example 3) at 4°C overnight or at room temperature for 1 hour. After removing the antigen by washing each well of the plate with PBST, the well was blocked with 250 μL of 0.1x TBS / 150 mM NaCl / 0.02% Skim Milk for at least 1 hour. The prepared culture supernatant was added to each well from which 0.1xTBS / 150mM NaCl / 0.02% Skim Milk had been removed. The plate was then allowed to stand at room temperature for 1 hour to allow the antibodies presented by the phages to bind to the antigens present in each well. After washing each well with 0.1xTBS / 150mM NaCl / 0.01% Tween20, HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech), diluted with 0.1xTBS / 150mM NaCl / 0.01% Tween20, was added and incubated for 1 hour. After washing with TBST, the color reaction of the solution in each well to which TMB single solution (ZYMED) had been added was stopped by the addition of sulfuric acid, and the color development was measured by absorbance at 450 nm. The results are shown in Figure 20. As shown in Figure 20, clones that bind to CD3 and human IgA were demonstrated, and by using the Dual Fab Library, it was possible to select clones that showed binding affinity to a second antigen (human IgA in Example 8).

[0260] (8-3) Binding of IgG with the obtained Fab domain to CD3 or human IgA For the clone shown to bind to CD3 and human IgA in (8-2), the VH fragment was amplified by PCR using a primer that specifically binds to the H chain of the Dual Fab Library from E. coli having that sequence. The amplified VH fragment was incorporated into an animal cell expression plasmid containing pE22Hh using the method of Reference Example 1, and expressed and purified as a one-arm antibody as in Example 6(6-2). The clone name and the sequence number of the H chain sequence are shown in Table 15. Specifically, the sequence obtained by linking the H chain shown in Table 15 with Kn010G3 (sequence number: 56) and the L chain with GLS3000 (sequence number: 53) and the Kappa sequence (sequence number: 55) was adopted, and expression and purification were performed according to Reference Example 1.

[0261] [Table 15]

[0262] The binding of the acquired antibody molecules containing the Fab region to CD3ε and human IgA was determined by electrochemiluminescence (ECL). Specifically, 25 μL each of a biotin-labeled CD3ε peptide (described in Example 7) or biotin-labeled human IgA (Reference Example 3), diluted in TBST solution (TaKaRa TBS with 0.1% Tween20), an antibody solution prepared to 2 μg / mL, and a sulfo-tag-attached anti-human IgG antibody (Invitrogen #628400) was added to each well of Nunc-Immuno(tm) MicroWell(tm) 96-well round plates (Nunc). After mixing, the mixtures were incubated at room temperature for at least 1 hour under light protection to form antibody-antigen complexes. 150 μL each of a TBST solution containing 0.5% BSA (referred to as blocking solution) was added to each well of a streptavidin plate (MSD) and incubated at room temperature for at least 1 hour. After removing the blocking solution, the plates were washed three times with 250 μL of TBS(-) solution. 50 μL of the antibody-antigen complex solution was added to each well and incubated at room temperature for 1 hour to conjugate the biotinylated antigen-antibody-detection sulfo-tag antibody complex solution to the streptavidin plate via the biotinylated antigen. After removing the antibody-antigen complex solution, the plates were washed three times with TBST solution, and 150 μL of 4xREAD buffer (MSD) diluted 2-fold with water was added to each well. The luminescence signal of the sulfo-tag was detected using a Sector Imager 2400 (MSD).

[0263] The results are shown in Figure 21. When clones that showed binding in phage ELISA were converted to IgG, including those containing some amino acid mutations, it was shown that the sequences that showed binding in phage ELISA still bound to CD3ε and human IgA even after being converted to IgG. These results demonstrate that antibodies that bind to a second antigen can be obtained from the Dual Fab Library. While panning using a typical phage library only yields the Fab domain, this library allows for the enrichment of clones that bind even when they become IgG containing the Fc region. Therefore, the Dual Fab Library can be described as a library that allows for the acquisition of Fab domains that possess the ability to bind to a second antigen while retaining their ability to bind to CD3.

[0264] (8-4) Evaluation of simultaneous binding of IgG containing the obtained Fab domain to CD3 (CD3ε) and human IgA. In (8-3), it was shown that clones obtained from the Dual Fab Library still exhibit binding even after becoming IgG. Next, it was determined whether the obtained IgG could simultaneously bind to CD3 (CD3ε) and human IgA using a competitive method (electrochemiluminescence (ECL)). If simultaneous binding occurs with CD3 (CD3ε) and human IgA, adding CD3 (CD3ε) to the antibody bound to IgA does not change the ECL signal. However, if simultaneous binding is not possible, adding CD3 (CD3ε) should cause some of the antibody to bind to CD3 (CD3ε), resulting in a decrease in the ECL signal.

[0265] Specifically, 25 μL of biotinylated human IgA diluted in TBST solution, 12.5 μL of antibody solution prepared to 1 μg / mL, 12.5 μL of TBST or CD3ε homodimer protein (9.4 pmol / μL) for competition, and 25 μL of sulfo-tag-tagged anti-human IgG antibody (Invitrogen #628400) were added to each well of Nunc-Immuno(tm) MicroWell(tm) 96-well round plates (Nunc), mixed, and incubated at room temperature for at least 1 hour under light protection to form antibody-antigen complexes. 150 μL of TBST solution containing 0.5% BSA (referred to as blocking solution; TBST solution is TBS from TaKaRa with 0.1% Tween20 added) was added to each well of a streptavidin plate (MSD) and incubated at room temperature for at least 1 hour. After removing the blocking solution, the plates were washed three times with 250 μL of TBST solution. 50 μL of the antibody-antigen complex solution was added to each well and incubated at room temperature for 1 hour to conjugate the biotinylated antigen-antibody-detection sulfo-tag antibody complex solution to the streptavidin plate via the biotinylated antigen. After removing the antibody-antigen complex solution, the plates were washed three times with TBST solution, and 150 μL of 4xREAD buffer (MSD) diluted twice with water was added to each well. The luminescence signal of the sulfo-tag was detected using a Sector Imager 2400 (MSD).

[0266] The results are shown in Figure 22. When CD3ε homodimer protein was added to induce competition, a decrease in the ECL signal was observed compared to when TBST was added. This result indicates that the molecule found in this study that binds to both CD3 and human IgA is a Dual Fab molecule that binds to CD3 but cannot bind to human IgA. This result indicates that antibodies with the ability to bind to a second antigen can be obtained from the Dual Fab library, and among them, it is possible to obtain Dual Fab molecules that cannot bind to multiple antigens simultaneously, such as those that bind to CD3 but cannot bind to the second antigen (or bind to the second antigen but cannot bind to CD3).

[0267] For those skilled in the art, it is obvious that if a binding molecule is found through the binding activity evaluation using phages as in (8-2), the variety of binding molecule sequences can be increased by increasing the number of evaluations. Therefore, it can be said that the Dual Fab Library is a library that can obtain Fab domains that have the ability to bind to a second antigen while retaining the ability to bind to CD3. Furthermore, although this embodiment used a Dual Fab Library with diversified H chains only, a larger library size (also called diversity, meaning that the library contains a variety of sequences) generally allows for the acquisition of more antigen-binding molecules. Therefore, a Dual Fab Library with diversified L chains can also be used to obtain Dual Fab molecules in the same way as shown in this embodiment.

[0268] If a Dual Fab molecule can be created as shown in Example 8, the Fab that binds to the third antigen and the antigen-binding domain can be identified using methods known to those skilled in the art, such as the hybridoma method or a method for selecting a bound antibody (or binding domain) from an antibody library. The antibody containing the identified antigen-binding domain (e.g., Fab) that binds to the third antigen and the Fab domain of the Dual Fab molecule can then be used to obtain a multispecific antibody using methods known to those skilled in the art, such as a method for creating antibodies with two different H chains by sharing the L chain (a technique for controlling the interface of each domain in the Fc region), the Cross Mab method, or the Fab Arm Exchange method. In other words, once the Dual Fab molecule is identified, the Fab that binds to the third antigen and the Dual Fab that binds to the first and second antigens shown in Example 8 can be combined using methods known to those skilled in the art to obtain a desired multispecific antibody.

[0269] (8-5) About the CD3 / Human IgA Dual Fab molecule Example 8 demonstrated that a Dual Fab molecule can be obtained that binds to CD3ε and human IgA, but does not bind to CD3ε and human IgA simultaneously. Furthermore, the addition of an antigen-binding domain that binds to a third antigen can be carried out by methods known to those skilled in the art. In recent years, it has been shown that IgA molecules modified to bind to EGFR, one of the cancer antigens, induce cell death in cancer cells expressing EGFR (J Immunol 2007; 179:2936-2943). As a mechanism, it has been reported that FcαR, an IgA receptor, is expressed in polymorphonuclear cells and induces autophagy in cancer cells (J Immunol 2011; 187:726-732). In this example, it has become clear that a Dual Fab molecule that binds to CD3 and IgA can be constructed, and if molecules that bind to FcαR via IgA are searched for using methods known to those skilled in the art (e.g., ELISA or ECL), an antitumor effect via FcαR can be expected. In other words, this Dual Fab can induce both T cell-mediated cytotoxicity via CD3ε binding and FcαR-mediated cytotoxicity via IgA binding in cells expressing any third antigen, thus promising strong cytotoxic activity.

[0270] [Example 9] Obtaining Fab domains that bind to CD3 and a second antigen (human CD154) from a Dual Fab Library (9-1) Obtaining a Fab domain that binds to human CD154 In Example 6, Fab domains (antibody fragments) that bind to human CD154 were identified from the Dual Fab library designed and constructed. Using biotin-labeled human CD154 as the antigen, antibody fragments capable of binding to human CD154 were enriched. Phage production was induced from E. coli containing the constructed phage display phagemide. A phage library solution was obtained by adding 2.5 M NaCl / 10% PEG to the culture medium of the phage-producing E. coli, precipitating the phage population, and then diluting the precipitate with TBS. Next, BSA was added to the phage library solution to a final concentration of 4% BSA. For panning, the common method of panning using antigens immobilized on magnetic beads was referenced (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). NeutrAvidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or Streptavidin-coated beads (Dynabeads M-280 Streptavidin) were used as magnetic beads. 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 antigen-phage complex was bound to the magnetic beads at room temperature for 15 minutes. The beads were washed three times with TBST (TBS containing 0.1% Tween20, manufactured by TaKaRa), and then washed two more times with 1 mL of TBS. Subsequently, 0.5 mL of 1 mg / mL of trypsin was added to the beads, and after being suspended at room temperature for 15 minutes, the beads were immediately separated using a magnetic stand, and the phage solution was recovered. The recovered phage solution was added to 10 mL of E. coli strain ER2738 in the logarithmic growth phase (OD600 0.4-0.5). The E. coli was slowly cultured with agitation at 37°C for 1 hour to infect the E. coli with the phages. Infected E. coli were seeded onto 225 mm x 225 mm plates. Next, a phage library was prepared by recovering phages from the culture medium of the seeded E. coli. This cycle, called panning, was repeated five times. For the second and subsequent panning cycles, the amount of human CD154 was set to 40 pmol.

[0271] (9-2) Binding of the Fab domain presented by the phage to CD3 or human CD154 From the single colonies of E. coli obtained by the method described above, the phage-containing culture supernatant was collected according to the standard method (Methods Mol. Biol. (2002) 178, 133-145). The phage-containing culture supernatant, to which BSA was added to a final concentration of 4% BSA, was subjected to ELISA according to the following procedure. StreptaWell 96 microtiter plates (Roche) were coated with 100 μL of PBS containing biotin-labeled antigens (biotin-labeled CD3ε peptide, biotin-labeled CD154) at 4°C overnight or at room temperature for 1 hour. After removing the antigens by washing each well of the plate with PBST, the wells were blocked with 250 μL of 0.1x TBS / 150 mM NaCl / 0.02% Skim Milk for at least 1 hour. The prepared culture supernatant was added to each well from which 0.1xTBS / 150mM NaCl / 0.02% Skim Milk had been removed. The plate was then allowed to stand at room temperature for 1 hour to allow the phage-presenting antibody to bind to the antigen present in each well. After washing each well with 0.1xTBS / 150mM NaCl / 0.01% Tween20, HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted with 0.1xTBS / 150mM NaCl / 0.01% Tween20 was added and incubated for 1 hour. After washing with TBST, the color reaction of the solution in each well to which TMB single solution (ZYMED) had been added was stopped by the addition of sulfuric acid, and the color development was measured by absorbance at 450 nm. The results are shown in Figure 23. As shown in Figure 23, clones that bind to CD3 and CD154 were demonstrated, and by using the Dual Fab Library, it was possible to select clones that bind to a second antigen (human CD154 in Example 9). Furthermore, as shown in Examples 7, 8, and 9, binding Fab domains could be obtained for three different antigens, demonstrating that the Dual Fab Library functions as a library for obtaining molecules that bind to a second antigen.

[0272] (9-3) Binding of IgG containing the acquired Fab domain to CD3 or human CD154 For the clone shown in (9-2) to bind to CD3 and human CD154, the VH fragment was amplified by PCR using a primer that specifically binds to the H chain of the Dual Fab Library from E. coli having that sequence. The amplified VH fragment was incorporated into an animal cell expression plasmid containing pE22Hh using the method of Reference Example 1, and expressed and purified as a one-arm antibody as in Example 6(6-2). The obtained sequence names and H chain sequence numbers are listed in Table 16. Specifically, the sequence obtained by ligating the H chain listed in Table 16 with Kn010G3 (Sequence ID: 56) and the L chain with GLS3000 (Sequence ID: 53) and the Kappa sequence (Sequence ID: 55) was adopted, and expression and purification were performed according to Reference Example 1.

[0273] [Table 16]

[0274] In (9-2), antibody molecules containing a Fab region, which were shown to bind to CD3 and human CD154 by phage ELISA, were tested by electrochemiluminescence (ECL) to determine whether they bound to CD3 and human CD154. Specifically, 25 μL of biotinylated CD3 or biotinylated human CD154 diluted in TBST solution, 25 μL of antibody solution prepared to 2 μg / mL, and 25 μL of sulfo-tag-added anti-human IgG antibody (Invitrogen #628400) were added to each well of Nunc-Immuno(tm) MicroWell(tm) 96-well round plates (Nunc), mixed, and incubated at room temperature for at least 1 hour under light shielding to form antibody-antigen complexes. 150 μL of TBST solution containing 0.5% BSA (referred to as blocking solution; TBST solution is TBS from TaKaRa with 0.1% Tween20 added) was added to each well of a streptavidin plate (MSD) and incubated at room temperature for at least 1 hour. After removing the blocking solution, the plates were washed three times with 250 μL of TBST solution. 50 μL of antibody-antigen complex solution was added to each well and incubated at room temperature for 1 hour to allow the biotinylated antigen-antibody-detection sulfo-tag antibody complex solution to bind to the streptavidin plate via the biotinylated antigen. After removing the antibody-antigen complex solution, the plates were washed three times with TBST solution, and 150 μL of 4x READ buffer (MSD) diluted twice with water was added to each well. The sulfo-tag luminescence signal was detected using a Sector Imager 2400 (MSD).

[0275] The results are shown in Figure 24. When clones that showed binding by phage ELISA were converted to IgG, including those containing some amino acid mutations, it was shown that the sequences that showed binding by phage ELISA still bound to CD3ε and human CD154 even after being converted to IgG. These results demonstrate that antibodies that bind to a second antigen can be obtained from the Dual Fab Library. While panning using a typical phage library only yields the Fab domain, this library shows that enrichment of clones that bind even when they include the Fc region in IgG is possible not only for human IgA but also for human CD154. Therefore, the Dual Fab Library can be described as a library that can obtain Fab domains that have the ability to bind to a second antigen while retaining their ability to bind to CD3.

[0276] (9-4) Evaluation of simultaneous binding of acquired Fab domain-containing IgG to CD3ε and human CD154 In (9-3), it was shown that clones obtained from the Dual Fab Library still exhibit binding even after becoming IgG. Next, it was determined whether the obtained IgG could simultaneously bind to CD3 and human CD154 using a competitive method (electrochemiluminescence (ECL)). If simultaneous binding occurs with CD3 and human CD154, adding CD3 to the antibody bound to CD154 does not change the ECL signal. However, if simultaneous binding is not possible, adding CD3 should cause some of the antibody to bind to CD3, resulting in a decrease in the ECL signal.

[0277] Specifically, 25 μL of biotinylated human CD154 diluted in TBST solution, 12.5 μL of antibody solution prepared to 1 μg / mL, 12.5 μL of TBST or CD3ε homodimer protein (9.4 pmol / μL) for competition, and 25 μL of sulfo-tag-tagged anti-human IgG antibody (Invitrogen #628400) were added to each well of Nunc-Immuno(tm) MicroWell(tm) 96-well round plates (Nunc), mixed, and incubated at room temperature for at least 1 hour under light protection to form antibody-antigen complexes. 150 μL of TBST solution containing 0.5% BSA (referred to as blocking solution; TBST solution is TBS from TaKaRa with 0.1% Tween20 added) was added to each well of a streptavidin plate (MSD) and incubated at room temperature for at least 1 hour. After removing the blocking solution, the plates were washed three times with 250 μL of TBST solution. 50 μL of the antibody-antigen complex solution was added to each well and incubated at room temperature for 1 hour to conjugate the biotinylated antigen-antibody-detection sulfo-tag antibody complex solution to the streptavidin plate via the biotinylated antigen. After removing the antibody-antigen complex solution, the plates were washed three times with TBST solution, and 150 μL of 4xREAD buffer (MSD) diluted twice with water was added to each well. The luminescence signal of the sulfo-tag was detected using a Sector Imager 2400 (MSD).

[0278] The results are shown in Figure 25. When CD3ε homodimer protein was added to induce competition, a decrease in the ECL signal was observed compared to when TBST was added. This result indicates that the molecule found in this study that binds to both CD3ε and human CD154 is a Dual Fab molecule that binds to CD3 but cannot bind to human CD154. This result indicates that antibodies that bind to a second antigen can be obtained from the Dual Fab library, and among them, it is possible to obtain Dual Fab molecules that cannot bind to multiple antigens simultaneously, such as those that bind to CD3 but cannot bind to the second antigen (or bind to the second antigen but cannot bind to CD3).

[0279] For those skilled in the art, it is obvious that if a binding molecule is found through the binding activity evaluation using phages as described in (9-2), the variety of binding molecule sequences can be increased by increasing the number of evaluations. Therefore, it can be said that the Dual Fab Library is a library that can obtain Fab domains that have the ability to bind to a second antigen while retaining the ability to bind to CD3. Furthermore, although this embodiment used a Dual Fab Library with only the H chain diversified, a larger library size (also called diversity, meaning that the library contains a variety of sequences) generally allows for the acquisition of more antigen-binding molecules. Therefore, a Dual Fab Library with diversified L chains can also be used to obtain Dual Fab molecules in the same way as shown in this embodiment.

[0280] If a Dual Fab molecule can be created as shown in Example 9, the Fab that binds to the third antigen and the antigen-binding domain can be identified using methods known to those skilled in the art, such as the hybridoma method or methods for selecting bound antibodies and antigen-binding domains from an antibody library. The identified antigen-binding domain (e.g., Fab) that binds to the third antigen and the antibody having the Fab domain of the Dual Fab molecule can then be used to obtain a multispecific antibody using methods known to those skilled in the art, such as a method for creating antibodies with two different H chains by sharing the L chain (a technique for controlling the interface of each domain in the Fc region), the Cross Mab method, or the Fab Arm Exchange method. In other words, once the Dual Fab molecule is identified, the Fab that binds to the third antigen and the Dual Fab that binds to the first and second antigens shown in Example 9 can be combined using methods known to those skilled in the art to obtain a desired multispecific antibody. The above examples demonstrate that by applying the Dual Fab library to various antigens, molecules that bind to CD3ε and a second antigen can be obtained. Furthermore, Examples 8 and 9 revealed that molecules that bind to both the first antigen (CD3ε) and the second antigen, but do not bind to both simultaneously, can be obtained. As mentioned above, identifying Fabs that bind to a third antigen is possible by methods known to those skilled in the art, and therefore, the desired antibody described in Example 1 can be obtained by using the Dual Fab library.

[0281] (9-5) About the CD3 / Human CD154 Dual Fab Molecules In Example 9, it was demonstrated that a Dual Fab molecule can be obtained that binds to CD3ε and human CD154, but does not bind to CD3ε and human CD154 simultaneously. Furthermore, the addition of an antigen-binding domain that binds to a third antigen can also be carried out by methods known to those skilled in the art. In recent years, it has been shown that agonist antibodies against CD40, the receptor for CD154, enhance antitumor activity in methods of transferring cancer antigen-reactive T cells (J Immunother. 2012 Apr;35(3):276-82.). In this example, it was revealed that a Dual Fab molecule that binds to both CD3 and CD154 can be constructed. By selecting an antibody that exhibits agonist activity against CD40 via CD154, an antitumor effect via CD40 can be expected. In other words, this Dual Fab is expected to enhance cytotoxic activity by T cells through binding to CD3ε and enhance the antitumor effect via CD40 agonist signaling through binding to CD154 in cells expressing any third antigen.

[0282] [Reference Examples] [Reference Example 1] Preparation of antibody expression vector and antibody expression and purification Amino acid substitutions were introduced using methods known to those skilled in the art, such as the QuikChange Site-Directed Mutagenesis Kit (Stratagene), PCR, or the Infusion Advantage PCR cloning kit (TAKARA), to construct the expression vector. The nucleotide sequence of the obtained expression vector was determined using methods known to those skilled in the art. The prepared plasmid was transiently introduced into human embryonic renal cell carcinoma cell line HEK293H (Invitrogen) or FreeStyle293 cells (Invitrogen), and antibody expression was performed. From the obtained culture supernatant, rProtein A Sepharose TM Antibodies were purified using Fast Flow (GE Healthcare) by methods known to those skilled in the art. The concentration of the purified antibody was calculated by measuring the absorbance at 280 nm using a spectrophotometer and using the extinction coefficient calculated from the obtained value by the PACE method (Protein Science 1995; 4: 2411-2423).

[0283] [Reference Example 2] Evaluation of antibody binding to ECM (Extracellular matrix) The antibody binding to the extracellular matrix (ECM) was evaluated using the following procedure, referencing WO2012093704A1. ECM Phenol red free (BD Matrigel #356237) was diluted to 2 mg / mL with TBS, and 5 μL was dropped into the center of each well of an ECL measurement plate (L15XB-3, MSD high bind) that had been chilled on ice. The plate was then sealed and left to stand overnight at 4°C. The plate with the ECM immobilized was returned to room temperature, and 150 μL of ECL Blocking Buffer (PBS with 0.5% BSA and 0.05% Tween 20) was added to each well. The plate was left to stand at room temperature for at least 2 hours or overnight at 4°C. Next, the antibody sample was diluted to 9 μg / mL using PBS-T (PBS with 0.05% Tween 20). The secondary antibody was diluted to 2 μg / mL with ECLDB (PBS with 0.1% BSA and 0.01% Tween 20). 20 μL of antibody solution and 30 μL of secondary antibody solution were added to a round-bottom plate containing 10 μL of ECLDB in each well, and the mixture was stirred at room temperature for 1 hour under light protection. The ECL Blocking Buffer was removed from an ECM plate containing ECL Blocking Buffer by inversion, and 50 μL of the aforementioned antibody / secondary antibody mixture was added to each well. The mixture was then allowed to stand at room temperature for 1 hour under light protection. After removing the samples by inversion, 150 μL of READ buffer (MSD) was added to each well, and the sulfo-tag emission signal was detected using a Sector Imager 2400 (MSD).

[0284] [Reference Example 3] Preparation of Human IgA The Fc portion of the naturally occurring human IgA sequence was used as the human IgA molecule (human IgA-Fc). To add biotin to the C-terminus of human IgA-Fc, a gene fragment encoding a specific sequence (AviTag sequence, SEQ ID NO: 79) to which biotin is added by a biotin ligase was linked via a linker. The gene fragment encoding the protein (SEQ ID NO: 80) in which human IgA-Fc and the AviTag sequence were linked was incorporated into an animal cell expression vector, and the constructed plasmid vector was introduced into FreeStyle293 cells (Invitrogen) using 293Fectin (Invitrogen). At this time, a gene expressing EBNA1 (SEQ ID NO: 81) and a gene expressing biotin ligase (BirA, SEQ ID NO: 82) were introduced simultaneously, and biotin was further added to biotin-label human IgA-Fc. Cells into which the gene had been introduced according to the procedure described above were cultured at 37°C and 8% CO2 for 6 days to allow the target protein to be secreted into the culture supernatant. The cell culture medium containing the target human IgA-Fc was filtered through a 0.22 μm bottle-top filter to obtain the culture supernatant. The culture supernatant, diluted with 20 mM Tris-HCl, pH 7.4, was applied to a HiTrap Q HP (GE Healthcare) column equilibrated with the same solution, and the target human IgA-Fc was eluted using a NaCl concentration gradient. Next, the HiTrap Q HP eluate, diluted with the same solution, was applied to a SoftLink Avidin column (Promega) equilibrated with 50 mM Tris-HCl, pH 8.0, and the target human IgA-Fc was eluted with 5 mM biotin, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0. Subsequently, unintended impurities, such as aggregates, were removed by gel filtration chromatography using Superdex200 (GE Healthcare), and purified human IgA-Fc was obtained with the buffer replaced with 20 mM Histidine-HCl, 150 mM NaCl, pH 6.0. [Industrial applicability]

[0285] The present invention makes it possible to enhance the activity generated by antigen-binding molecules, and also makes it possible to avoid cross-linking between different cells caused by binding to antigens expressed on different cells, which is thought to be a cause of side effects, thereby providing a polypeptide suitable for use as a pharmaceutical.

Claims

1. Antigen-binding molecules including the following: (a) An antibody variable region that can bind to a first antigen which is CD3 and to a second antigen which is a non-CD3 molecule; Here, the second antigen is FcγR, TLR, lectin, IgA, immune checkpoint molecule, TNF superfamily molecule, TNFR superfamily molecule, or NK receptor molecule. The antibody variable region includes an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH) that include binding regions for the first antigen and the second antigen. The antibody heavy chain variable domain has at least one amino acid modification using the sequence described in Sequence ID No. 13 as a template sequence. The amino acid modification includes substitution of the Kabat numbering 52c-54 of the heavy chain variable domain of the template sequence with an amino acid sequence that binds to the second antigen. The number of substituted amino acids is one or more and five or less; and (b) an antibody variable region that binds to a third antigen different from the first antigen and the second antigen; Here, the antigen-binding molecule is one in which all three amino acids (SNN) of Kabat numbering 52c-54 are replaced with an amino acid sequence that binds to a second antigen.

2. The antigen-binding molecule according to claim 1(a), wherein the antibody variable region of claim 1(a) does not bind to the first antigen when bound to the second antigen, and does not bind to the second antigen when bound to the first antigen.

3. The antigen-binding molecule according to claim 1 or 2, wherein both the binding region for a first antigen and the binding region for a second antigen are located within the same antibody variable region.

4. The antigen-binding molecule according to any one of claims 1 to 3, further comprising an antibody Fc region.

5. The antigen-binding molecule according to claim 4, wherein the Fc region is an Fc region having reduced binding activity to FcγR compared to the Fc region of a natural human IgG1 antibody.

6. An antigen-binding molecule according to any one of claims 1 to 5, which is a multispecific antibody.

7. The antigen-binding molecule according to any one of claims 1 to 6, wherein the third antigen is a molecule specifically expressed in cancer tissue.

8. A pharmaceutical composition comprising an antigen-binding molecule according to any one of claims 1 to 7 and a medically acceptable carrier.

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