Antigen-binding molecules containing altered antibody variable regions

Abstract

The present inventors have successfully produced an antigen-binding molecule, which comprises an antibody variable region that has binding activity to a molecule expressed on the surface of a T cell and a molecule expressed on the surface of other immune cells, but does not bind simultaneously. The present invention can produce an antigen-binding molecule that can avoid the side effects produced when T cells and other immune cells are cross-linked, and can further provide an antigen-binding molecule suitable as a pharmaceutical.

Description

[0001] This application is a divisional application of a patent application with an application date of November 11, 2014, international application number PCT / JP2014 / 079785, Chinese application number 201480072799.3, and invention name “Induction of pluripotent cells”. Technical Field

[0002] The present invention provides an antigen-binding molecule, which comprises: an antibody variable region that can bind to two different antigens (a first antigen and a second antigen) but not to both antigens simultaneously, and an antibody variable region that binds to a third antigen different from these antigens; a pharmaceutical composition containing the antigen-binding molecule; and methods for preparing the same. Background Art

[0003] Antibodies are highly stable in plasma and have few side effects, so they have attracted attention as pharmaceuticals (Nat. Biotechnol. (2005) 23, 1073-1078 (Non-patent document 1) and Eur J Pharm Biopharm. (2005) 59 (3), 389-396 (Non-patent document 2)). Antibodies not only induce antigen-binding effects, agonist effects, or antagonist effects, but also induce cytotoxic activities (also called effector functions) based on effector cells, such as ADCC (Antibody Dependent Cytotoxicity), ADCP (Antibody Dependent Cell phagocytosis), and CDC (Complement Dependent Cellular Toxicity). In particular, since antibodies of the IgG1 subclass exert effector functions on cancer cells, a variety of antibody pharmaceuticals have been developed in the field of cancer.

[0004] In order for an antibody to express ADCC, ADCP, and CDC, it is necessary to bind the Fc region of the antibody to antibody receptors (FcγR) and various complement components present in effector cells such as NK cells or macrophages. For humans, as a protein family of FcγR, isotypes of FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb have been reported, and their respective allotypes have also been reported (Immunol. Lett. (2002) 82, 57-65 (Non-patent Document 3)). Among these isotypes, FcγRIa, FcγRIIa, and FcγRIIIa have a domain called ITAM (Immunoreceptor Tyrosine-based Activation Motif) in the intracellular domain to transmit activation signals. On the other hand, only FcγRIIb has a domain called ITIM (Immunoreceptor Tyrosine-based Inhibitory Motif) in the intracellular domain to transmit inhibitory signals. It is also known that any FcγR can be cross-linked through immune complexes, etc., to transmit signals (Nat. Rev. Immunol. (2008) 8, 34-47 (Non-patent Document 4)). In fact, when antibodies exert effector functions on cancer cells, FcγRs on the effector cell membranes are clustered in the Fc regions of multiple antibodies bound to the cancer cell membrane, and activation signals are transmitted through the effector cells. As a result, although the cell-killing effect can be exerted, the cross-linking of FcγRs at this time is limited to effector cells present near cancer cells, so it is shown that immune activation only occurs locally in cancer cells (Ann. Rev. Immunol. (1988). 6. 251-81 (Non-patent Document 5)).

[0005] Natural immunoglobulins bind to antigens in the variable region and to receptors such as FcγR, FcRn, FcαR, FcεR, or complement in the constant region. It has been reported that one of the binding molecules that interacts with the Fc region of IgG is FcRn, and each molecule binds to each heavy chain of the antibody, so 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). In addition, it has been shown that several amino acid residues in the hinge region and CH2 domain of the antibody and the sugar chain added to Asn at position 297 in the EU numbering that binds to the CH2 domain are important for the binding of FcγR to the Fc region of the antibody (Chem. Immunol. (1997), 65, 88-110 (Non-patent Document 6), Eur. J. Immunol. (1993) 23, 1098-1104 (Non-patent Document 7), Immunol. (1995) 86, 319-324 (Non-patent Document 8)). Various Fc region mutants having FcγR binding properties have been studied around this binding site, and Fc region mutants having higher binding activity to activated Fcγ receptors have been obtained (WO2000 / 042072 (Patent Document 1), WO2006 / 019447 (Patent Document 2)). For example, Lazar et al. succeeded in increasing the binding activity of human IgG1 to human FcγRIIIa (V158) by about 370 times by replacing Ser at position 239, Ala at position 330, and Ile at position 332 of EU numbering of human IgG1 with Asp, Leu, and Glu, respectively (Proc. Natl. Acad. Sci. USA (2006) 103, 4005-4010 (Non-patent Document 9), WO2006 / 019447 (Patent Document 2)). The ratio of the binding activity of this variant to FcγRIIIa and FcγRIIb (A / I ratio) was about 9 times that of the native type. In addition, Shinkawa et al. succeeded in increasing the binding activity to FcγRIIIa by about 100 times by deleting the fucose of the sugar chain added to Asn at EU numbering position 297 (J. Biol. Chem. (2003) 278, 3466-3473 (Non-patent Document 10)). By these methods, the ADCC activity of human IgG1 can be greatly improved compared to native human IgG1.

[0006] Conventional natural IgG antibodies recognize and bind to one epitope through their variable region (Fab), so they can only bind to one antigen. On the other hand, in cancer or inflammation, it is known that multiple proteins are involved, and there may be crosstalk between proteins. For example, in immune diseases, it is known that several inflammatory cytokines (TNF, IL1 or IL6) are involved (Nat. Biotech., (2011) 28, 502-10 (non-patent document 11)). It is also known that one of the mechanisms for acquiring drug resistance in cancer is the activation of other receptors (Endocr Relat Cancer (2006) 13, 45-51 (non-patent document 12)). In such a case, it is impossible to inhibit multiple proteins in conventional antibodies that recognize one epitope.

[0007] As molecules that inhibit multiple targets, antibodies that bind to two or more antigens with one molecule (called bispecific antibodies) have been studied. By improving natural IgG antibodies, it is possible to impart binding activity to two different antigens (the first antigen and the second antigen) (MAbs. (2012) Mar 1, 4 (2)). Therefore, it not only has the effect of neutralizing two or more antigens with one molecule, but also has the effect of enhancing anti-tumor activity by cross-linking cells with cytotoxic activity with cancer cells. So far, as molecular forms of bispecific antibodies, the following have been reported: molecules with an antigen binding site added to the N-terminus or C-terminus of an antibody (DVD-Ig or scFv-IgG), or molecules with two Fab regions of an antibody having different sequences (bispecific antibodies with a common L chain and hybridized hybridomas), molecules with one Fab region recognizing two antigens (Two-in-one IgG), and molecules with a loop site in the CH3 region as a new antigen binding site (Fcab) (Nat. Rev. (2010), 10, 301-316 (Non-patent Document 13), Peds (2010), 23 (4), 289-297 (Non-patent Document 14)). Since any bispecific antibody interacts with FcγR in the Fc region, the effector function of the antibody is preserved. Therefore, any antigen recognized by the bispecific antibody also binds to FcγR at the same time, and exhibits ADCC activity against cells expressing the antigen.

[0008] As long as the antigens recognized by the bispecific antibody are all cancer-specific antigens, binding to any antigen will show cytotoxic activity to cancer cells, so it can be expected to have a more effective anti-cancer effect than the usual antibody pharmaceuticals that recognize one antigen. However, among the antigens recognized by the bispecific antibody, any one antigen, when expressed in normal tissue or in cells expressed in immune cells, will also cause damage to normal tissue or release of cytokines due to cross-linking with FcγR (J.Immunol. (1999) Aug 1, 163 (3), 1246-52 (non-patent literature 15)). As a result, stronger side effects are induced.

[0009] For example, Catumaxomab is known as a bispecific antibody that recognizes a protein expressed on T cells and a protein expressed on cancer cells (cancer antigen). Catumaxomab binds to a cancer antigen (EpCAM) and a CD3ε chain expressed on T cells in two Fabs, respectively. Catumaxomab induces T cell-mediated cytotoxic activity by binding to cancer antigens and CD3ε simultaneously, and induces cytotoxic activity mediated by antigen-presenting cells such as NK cells or macrophages by binding to cancer antigens and FcγR simultaneously. By utilizing the two cytotoxic activities, Catumaxomab has shown a high therapeutic effect on malignant ascites by intraperitoneal administration, and has been recognized in Europe (Cancer Treat Rev. (2010) Oct 36 (6), 458-67 (Non-patent Document 16)). Furthermore, it has been reported that an example of an antibody that reacts to cancer cells has been generated by administering Catumaxomab, and it has been clarified that acquired immunity has been induced (Future Oncol. (2012) Jan 8 (1), 73-85 (Non-patent Document 17)). Based on the results, antibodies that possess both T cell-mediated cytotoxic activity and cell-mediated action on NK cells, macrophages, etc. via FcγR (particularly called trifunctional antibodies) are expected to have strong anti-tumor effects and induce adaptive immunity, and therefore have attracted attention.

[0010] However, trifunctional antibodies bind to CD3ε and FcγR simultaneously even in the absence of cancer antigens, so even in the absence of cancer cells, T cells expressing CD3ε and cells expressing FcγR cross-link and produce a large amount of various cytokines. Due to the induction of the production of various cytokines independent of such cancer antigens, the administration of trifunctional antibodies is currently limited to the peritoneal cavity (Cancer Treat Rev. 2010 Oct 36 (6), 458-67 (Non-patent Document 16)), and systemic administration is extremely difficult due to severe cytokine storm-like side effects (Cancer Immunol Immunother. 2007 Sep; 56 (9): 1397-406 (Non-patent Document 18)).

[0011] Furthermore, in conventional bispecific antibodies, both the cancer antigen (EpCAM) as the first antigen and CD3ε as the second antigen can bind to FcγR simultaneously, and therefore it is impossible to avoid the side effects caused by the simultaneous binding of FcγR and the second antigen CD3ε from a molecular structure perspective.

[0012] In recent years, improved antibodies that induce T cell-mediated cytotoxic activity while avoiding side effects have been provided by using an Fc region with reduced binding activity to FcγR (WO2012 / 073985).

[0013] However, even such antibodies cannot bind to cancer antigens and act on two immune receptors, CD3ε and FcγR, at the same time due to their molecular structure.

[0014] To date, no antibodies have been known that can exert both T cell-mediated cytotoxic activity and cytotoxic activity mediated by cells other than T cells specifically for cancer antigens while avoiding side effects.

[0015] Prior art literature

[0016] Patent Literature

[0017] Patent Document 1: WO2000 / 042072

[0018] Patent Document 2: WO2006 / 019447

[0019] Non-patent literature

[0020] Non-patent document 1: Nat. Biotechnol. (2005) 23, 1073-1078

[0021] Non-patent document 2: Eur J Pharm Biopharm. (2005) 59(3), 389-396

[0022] Non-patent document 3: Immunol. Lett. (2002) 82, 57-65

[0023] Non-patent document 4: Nat. Rev. Immunol. (2008) 8, 34-47

[0024] Non-patent document 5: Ann. Rev. Immunol. (1988). 6. 251-81

[0025] Non-patent document 6: Chem. Immunol. (1997), 65, 88-110

[0026] Non-patent document 7: Eur. J. Immunol. (1993) 23, 1098-1104

[0027] Non-patent document 8: Immunol. (1995) 86, 319-324

[0028] Non-patent document 9: Proc. Natl. Acad. Sci. USA (2006) 103, 4005-4010

[0029] Non-patent document 10: J. Biol. Chem. (2003) 278, 3466-3473

[0030] Non-patent document 11: Nat. Biotech., (2011) 28, 502-10

[0031] Non-patent document 12: Endocr Relat Cancer (2006) 13, 45-51

[0032] Non-patent document 13: Nat. Rev. (2010), 10, 301-316

[0033] Non-patent document 14: Peds (2010), 23(4), 289-297

[0034] Non-patent document 15: J. Immunol. (1999) Aug 1, 163(3), 1246-52

[0035] Non-patent document 16: Cancer Treat Rev. (2010) Oct 36(6), 458-67

[0036] Non-patent literature 17: Future Oncol. (2012) Jan 8(1), 73-85

[0037] Non-patent document 18: Cancer Immunol Immunother. 2007 Sep; 56(9): 1397-406. Summary of the invention

[0038] Problems to be solved by the invention

[0039] The present invention has been completed based on the above situation, and its subject is to provide an antigen-binding molecule, which comprises: an antibody variable region having a binding activity for two different antigens (a first antigen and a second antigen) but not binding to these antigens at the same time, and a variable region binding to an antigen different from these antigens (a third antigen); a pharmaceutical composition containing the antigen-binding molecule; and a method for preparing the antigen-binding molecule.

[0040] Means for solving problems

[0041] The inventors have conducted in-depth research to solve the above-mentioned problems. As a result, the inventors have produced an antigen-binding molecule, which comprises an antibody variable region that has binding activity for two different antigens (the first antigen and the second antigen) but does not bind to these antigens at the same time, and a variable region that binds to an antigen (the third antigen) different from these antigens, and by utilizing the binding activity of the antigen-binding molecule for three different antigens, the activity generated by the antigen-binding molecule is successfully enhanced. Furthermore, an antigen-binding molecule has been successfully produced, which can avoid the cross-linking between the different cells that is considered to be the cause of side effects when the existing multispecific antigen-binding molecule is used as a pharmaceutical product, which is produced by binding to the antigen expressed on different cells.

[0042] More specifically, the present invention relates to the following.

[0043] [1] An antigen-binding molecule comprising:

[0044] An antibody variable region that can bind to a first antigen and a second antigen different from the first antigen, but does not bind to both the first antigen and the second antigen simultaneously; and

[0045] A variable region that binds to a third antigen that is different from the first antigen and the second antigen.

[0046] [2] An antigen-binding molecule comprising an antibody variable region, wherein the amino acids in the heavy chain variable region of the antibody variable region are altered so as to bind to a first antigen and a second antigen different from the first antigen, but not to bind to both the first antigen and the second antigen simultaneously.

[0047] [3] The antigen-binding molecule of [1] or [2], wherein the variable region that does not bind to both the first antigen and the second antigen simultaneously is a variable region that does not bind to both the first antigen and the second antigen simultaneously and is expressed on different cells.

[0048] [4] The antigen-binding molecule according to any one of [1] to [3], further comprising an antibody Fc region.

[0049] [5] The antigen-binding molecule of [4], wherein the Fc region has reduced FcγR-binding activity compared to the Fc region of a native human IgG1 antibody.

[0050] [6] The antigen-binding molecule according to any one of [1] to [5], wherein the antigen-binding molecule is a multispecific antibody.

[0051] [7] The antigen-binding molecule according to any one of [1] to [6], wherein the antibody variable region capable of binding to the first antigen and the second antigen is a variable region in which at least one amino acid modification has been introduced.

[0052] [8] The antigen-binding molecule of [7], wherein the alteration is substitution or insertion of at least one amino acid.

[0053] [9] The antigen-binding molecule of [7] or [8], wherein the alteration is substitution of a portion of the amino acid sequence of the variable region that binds to the first antigen with an amino acid sequence that binds to the second antigen, or insertion of an amino acid sequence that binds to the second antigen into the amino acid sequence of the variable region that binds to the first antigen.

[0054]

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

[0055]

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

[10] , wherein the amino acids to be modified are amino acids in the CDR1, CDR2, CDR3, or FR3 region of an antibody variable region.

[0056]

[12] The antigen-binding molecule of any one of [7] to

[11] , wherein the amino acids to be altered are amino acids in the loop region.

[0057]

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

[11] , wherein the amino acid to be modified is at least one amino acid selected from the group consisting of amino acids with Kabat numbers 31 to 35, 50 to 65, 71 to 74, and 95 to 102 in the H chain variable region and amino acids with Kabat numbers 24 to 34, 50 to 56, and 89 to 97 in the L chain variable region of the antibody.

[0058]

[14] The 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 T cells, and the other antigen is a molecule expressed on the surface of T cells or other immune cells.

[0059]

[15] The antigen-binding molecule of

[14] , wherein either the first antigen or the second antigen is CD3, and the other antigen is FcγR, TLR, lectin, IgA, an immune restriction site molecule, a TNF superfamily molecule, a TNFR superfamily molecule, or a NK receptor molecule.

[0060]

[16] The antigen-binding molecule of

[14] or

[15] , wherein the third antigen is a molecule specifically expressed in cancer tissue.

[0061]

[17] A pharmaceutical composition comprising the antigen-binding molecule of any one of [1] to

[16] and a medically acceptable carrier.

[0062]

[18] A method for producing the antigen-binding molecule of any one of [1] to

[16] , comprising the following steps (i) to (iv):

[0063] Step (i): preparing a library of antigen-binding molecules, wherein at least one amino acid in the variable region of an antibody binding to a first antigen or a second antigen is changed, and the library comprises variable regions in which at least one amino acid in the changed variable region is different from each other;

[0064] Step (ii): selecting, from the prepared library, an antigen-binding molecule comprising a variable region that has binding activity to the first antigen and the second antigen but does not bind to the first antigen and the second antigen simultaneously;

[0065] Step (iii): culturing a host cell 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, so that the antigen-binding molecule containing the variable region of an antibody that binds to the first antigen and the second antigen but not to the first antigen and the second antigen simultaneously, and / or the variable region that binds to the third antigen is expressed; and

[0066] Step (iv): recovering the antigen-binding molecules from the above host cell culture.

[0067]

[19] The preparation method described in

[18] , wherein the variable region contained in the antigen-binding molecule selected in step (ii) that does not bind to the first antigen and the second antigen at the same time is a variable region that does not bind to the first antigen and the second antigen at the same time that are expressed on different cells, respectively.

[0068]

[20] The method of

[18] or

[19] , wherein the host cells cultured in step (iii) further contain a nucleic acid encoding an antibody Fc region.

[0069]

[21] The method of

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

[0070]

[22] The method of any one of

[18] to

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

[0071]

[23] The method according to any one of

[18] to

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

[0072]

[24] The preparation method described in

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

[0073]

[25] The method according to any one of

[18] to

[24] , wherein the alteration refers to an amino acid alteration in the CDR1, CDR2, CDR3 or FR3 region of the antibody variable region.

[0074]

[26] The method according to any one of

[18] to

[25] , wherein the alteration refers to an alteration of amino acids in the loop region.

[0075]

[27] The method of any one of

[18] to

[25] , wherein the alteration refers to the alteration of at least one amino acid selected from Kabat numbers 31 to 35, 50 to 65, 71 to 74 and 95 to 102 in the antibody H chain variable region and Kabat numbers 24 to 34, 50 to 56 and 89 to 97 in the L chain variable region.

[0076]

[28] The method of any one of

[18] to

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

[0077]

[29]

[28] The preparation method described in, wherein either the first antigen or the second antigen is CD3, and the other antigen is FcγR, TLR, IgA, lectin, immune restriction point molecule, TNF superfamily molecule, TNFR superfamily molecule or NK receptor molecule.

[0078]

[30] The method of

[28] or

[29] , wherein the third antigen is a molecule specifically expressed by cancer tissue.

[0079]

[31] A method for treating cancer, comprising administering the antigen-binding molecule of any one of [1] to

[16] .

[0080]

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

[16] for use in treating cancer.

[0081]

[33] Use of the antigen-binding molecule of any one of [1] to

[16] for the preparation of a therapeutic drug for cancer.

[0082]

[34] A method for producing a therapeutic drug for cancer, comprising the step of using the antigen-binding molecule of any one of [1] to

[16] .

[0083]

[35] Any solution obtained by combining one or more of the above-mentioned solutions is also included in the present invention as long as there is no technical contradiction based on the technical common sense of those skilled in the art. This is of course understandable to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 : This is a conceptual diagram of an antibody that binds to a first antigen and a second antigen, but not simultaneously.

[0085] Figure 2 : This is a conceptual diagram of an antibody that does not cause cross-linking because it does not bind to two antigens at the same time.

[0086] Figure 3 : This is a conceptual diagram of an antibody that does not bind to two cells at the same time even if it binds to two antigens at the same time.

[0087] Figure 4 : A conceptual diagram of an antibody that cross-links cancer cells and T cells expressing the first receptor.

[0088] Figure 5 : A conceptual diagram of an antibody that cross-links cancer cells and cells expressing a second receptor.

[0089] Figure 6 : A conceptual diagram of an antibody that cross-links cancer cells and immune cells but does not cross-link immune cells with each other.

[0090] Figure 7 : A diagram showing the results of cellular ELISA of CE115 against CD3ε.

[0091] Figure 8 : A diagram showing the molecular form of EGFR_ERY22_CE115.

[0092] Fig. 9 : It is a figure showing the TDCC result of EGFR_ERY22_CE115 (SK-pca13a).

[0093] Fig.10 : A diagram showing the binding activity of humanized CE115 to CD3ε.

[0094] Fig.11 : A diagram showing the results of ECL-ELISA for detecting the binding of RGD-inserted CE115 to integrin.

[0095] Fig.12 : A diagram showing the results of ECL-ELISA for detecting the binding of RGD-inserted CE115 to CD3ε.

[0096] Fig.13 : This is a diagram showing the results of ECL-ELSIA for detecting the simultaneous binding of RGD-inserted CE115 to integrins and CD3ε. The results of variants for simultaneous binding are shown.

[0097] Fig.14 : A diagram showing the results of ECL-ELISA for simultaneous binding of RGD to CE115. The results of variants for which simultaneous binding is not performed are shown.

[0098] Fig.15 : A diagram showing the results of ECL-ELISA for detecting the binding of TLR2-binding peptide-inserted CE115 to TLR2.

[0099] Fig.16 : A diagram showing the results of ECL-ELISA for detecting the binding of TLR2-binding peptide inserted into CE115 to CD3ε.

[0100] Fig.17 : A diagram showing the results of ECL-ELISA for detecting the simultaneous binding of the TLR2-binding peptide inserted into CE115 to TLR2 and CD3.

[0101] Fig.18 : This is an example of a sensorgram of an antibody with a binding ratio of less than 0.8. The vertical axis represents RU value (response) and the horizontal axis represents time.

[0102] Fig.19 : A diagram showing the binding of the Fab domain presented by the phage to CD3ε and IL6R.

[0103] Fig. 20 : is a diagram showing 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.

[0104] Fig.21 : A diagram showing the binding of IgG-converted clones to CD3ε and human IgA (hIgA).

[0105] Fig. 22 : A diagram showing that the binding of the IgG-converted clone to human IgA is inhibited by CD3ε, and thus the clone cannot bind to human IgA (hIgA) and CD3ε simultaneously.

[0106] Fig.23 : is a diagram showing the binding of the Fab domain presented by the phage to CD3ε and human CD154. NC indicates binding to a plate without antigen immobilization.

[0107] Fig.24 : A diagram showing the binding of IgG-converted clones to CD3ε and human CD154.

[0108] Fig.25 : A diagram showing that the binding of the IgG-converted clone to human CD154 is inhibited by CD3ε, and thus the clone cannot bind to both human CD154 and CD3ε simultaneously. DETAILED DESCRIPTION

[0109] In the present invention, "antibody variable region" refers to a region generally composed of four framework regions (FR) and three complementarity determining regions (CDR) sandwiched therein, and also includes partial sequences thereof as long as it has the activity of binding to part or all of the antigen. Regions containing antibody light chain variable regions (VL) and antibody heavy chain variable regions (VH) are particularly preferred. The antibody variable region of the present invention can be any sequence, such as mouse antibodies, rat antibodies, rabbit antibodies, goat antibodies, camel antibodies, and humanized antibodies obtained by humanizing these non-human antibodies, as well as human antibodies, and can be antibody variable regions of any origin. "Humanized antibodies" are also called reshaped human antibodies, and refer to antibodies obtained by transplanting the complementarity determining regions (CDRs; complementarity determining regions) of antibodies derived from mammals other than humans, such as mouse antibodies, into the CDRs of human antibodies. The method for identifying CDR is well known (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al., Nature (1989) 342: 877). In addition, the general gene recombination method is also well known (see European Patent Application Publication No. EP125023 and WO96 / 02576).

[0110] The "antibody variable region does not bind to the first antigen and the second antigen at the same time" of the present invention means that the antibody variable region of the present invention cannot bind to the second antigen in the state of binding to the first antigen, and on the contrary, the variable region cannot bind to the first antigen in the state of binding to the second antigen. Here, in "not binding to the first antigen and the second antigen at the same time", it also includes that the two cells of the first antigen-expressing cell and the second antigen-expressing cell are not cross-linked, or the first antigen and the second antigen expressed by each cell are not combined at the same time. Further, the following situation is also included: when the first antigen and the second antigen are not expressed on the cell membrane like soluble proteins, or when both parties are present on the same cell, they can be combined with both parties of the first antigen and the second antigen at the same time, but when expressed on different cells, they cannot be combined at the same time. As such an antibody variable region, as long as it has this function, it is not particularly limited, for example, it can be cited: a part of the amino acids in the variable region of the IgG type antibody is changed into a variable region that binds to the desired antigen. As the amino acids to be modified, for example, amino acids in the variable region of an antibody that binds to the first antigen or the second antigen can be selected that do not lose the binding to the antigen by amino acid modification.

[0111] Here, "expressed on different cells" only needs to be expressed on separate cells, and such a cell combination may be, for example, cells of the same type such as T cells and other T cells, or cells of different types such as T cells and NK cells.

[0112] The amino acid modifications of the present invention may be used alone or in combination of two or more.

[0113] When multiple combinations are used, the number of combinations is not particularly limited and can be appropriately set within the scope that can achieve the purpose of the invention, for example, more than 2 and less than 30, preferably more than 2 and less than 25, more than 2 and less than 22, more than 2 and less than 20, more than 2 and less than 15, more than 2 and less than 10, more than 2 and less than 5, more than 2 and less than 3.

[0114] In the case of multiple combinations, the amino acid changes may be added only to the heavy chain variable region or the light chain variable region of the antibody, or the amino acid changes may be added to both the heavy chain variable region and the light chain variable region in an appropriately distributed manner.

[0115] As long as the amino acid residues to be changed maintain their antigen binding activity, changes in one or more amino acid residues in the variable region can be accepted. When changing the amino acids in the variable region, there is no particular limitation, but it is preferred to maintain the binding activity of the antibody before the change, for example, compared with before the change, it is preferably 50% or more, more preferably 80% or more, and further preferably 100% or more of the binding activity. Moreover, by changing the amino acids, the binding activity can be improved, for example, the binding activity can be 2 times, 5 times, 10 times, etc. compared with before the change.

[0116] As preferred regions for amino acid changes, there can be mentioned: solvent exposed regions and loop regions in the variable region. Among them, CDR1, CDR2, CDR3, FR3 regions, and loop regions are preferred. Specifically, Kabat numbers 31 to 35, 50 to 65, 71 to 74, 95 to 102 of the H chain variable region, and Kabat numbers 24 to 34, 50 to 56, and 89 to 97 of the L chain variable region are preferred, and Kabat numbers 31, 52a to 61, 71 to 74, 97 to 101 of the H chain variable region, and Kabat numbers 24 to 34, 51 to 56, and 89 to 96 of the L chain variable region are more preferred. In addition, when amino acids are changed, amino acid combinations that improve the binding activity to antigens can be introduced.

[0117] It should be noted that, in the present invention, the "loop region" refers to a region where residues not involved in maintaining the β-barrel structure of an immunoglobulin are present.

[0118] In the present invention, the change of amino acids refers to any one of substitution, deletion, addition, insertion, or modification, or a combination thereof. In the present invention, the change of amino acids may be referred to as amino acid mutation, and is used with the same meaning.

[0119] When substituting an amino acid residue, the purpose is to modify the following points (a) to (c), for example, by substituting with another amino acid residue.

[0120] (a) The main chain structure of the polypeptide in the region of the folded structure or the helical structure;

[0121] (b) charge or hydrophobicity in the target site; or

[0122] (c) Size of the side chain.

[0123] Amino acid residues are generally classified into the following groups based on the properties of their side chains:

[0124] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0125] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0126] (3) Acidic: Asp, Glu;

[0127] (4) Basic: His, Lys, Arg;

[0128] (5) Residues that affect chain orientation: Gly, Pro; and

[0129] (6) Aromaticity: Trp, Tyr, Phe.

[0130] Such substitutions of amino acid residues within each group are referred to as conservative substitutions, whereas substitutions of amino acid residues between other groups are referred to as non-conservative substitutions.

[0131] The substitution in the present invention may be a conservative substitution, a non-conservative substitution, or a combination of a conservative substitution and a non-conservative substitution.

[0132] In addition, the addition of changes in amino acid residues also includes the following situations: in the variable region of an antibody that binds to the first antigen or the second antigen, a variable region that can bind to the first antigen and the second antigen but cannot bind simultaneously is selected from the amino acids that are randomly changed by amino acid changes without losing the amino acid binding to the antigen, or a peptide known to have binding activity to the desired antigen is inserted into the above-mentioned region. As peptides known to have binding activity to the desired antigen in advance, for example, the peptides shown in Table 1 can be cited.

[0133] [Table 1]

[0134]

[0135] As one embodiment of the present invention, an antigen-binding molecule is provided, the antigen-binding molecule comprising: an antibody variable region that can bind to a first antigen and a second antigen different from the first antigen, but does not bind to the first antigen and the second antigen at the same time, and changes the amino acid of the heavy chain variable region. For example, by introducing the above-mentioned amino acid changes (any one of substitution, deletion, addition, insertion, or modification, or a combination thereof) into the heavy chain variable region, an antibody variable region that can bind to the first antigen and the second antigen different from the first antigen, but does not bind to the first antigen and the second antigen at the same time, can be prepared. The position where the amino acid change is introduced is preferably the heavy chain variable region, and as a more preferred region, it can be cited: the solvent-exposed region and the loop region in the variable region. Among them, CDR1, CDR2, CDR3, FR3 region, loop region are preferred. Specifically, Kabat numbers 31 to 35, 50 to 65, 71 to 74, 95 to 102 of the H chain variable region are preferred, and Kabat numbers 31, 52a to 61, 71 to 74, 97 to 101 of the H chain variable region are more preferred. Furthermore, when amino acids are modified, amino acids that enhance the antigen-binding activity may be introduced in combination.

[0136] In the antibody variable region of the present invention, in addition to the above-mentioned changes, known changes can also be combined. For example, the modification of the N-terminal glutamine of the variable region to pyroglutamic acid by pyroglutamylation is a modification well known to those skilled in the art. Therefore, the antibody of the present invention, when the N-terminal of its heavy chain is glutamine, comprises a variable region in which glutamine is modified to pyroglutamic acid.

[0137] In addition, for example, the variable regions of these antibodies may be further modified with amino acids in order to improve binding to antigens, pharmacokinetics, stability, and antigenicity. The variable regions of the antibodies of the present invention may be modified to have pH-dependent binding to antigens, thereby repeatedly binding to antigens (WO / 2009 / 125825).

[0138] Furthermore, for example, amino acid changes that change the antigen-binding activity depending on the concentration of the target tissue-specific compound may be added to the variable regions of these antibodies that bind to the third antigen (WO2013 / 180200).

[0139] Furthermore, for example, the variable region may be altered for the following purposes: improving binding activity, improving specificity, lowering pI, imparting pH-dependent properties to antigen binding, improving binding thermal stability, improving solubility, stability to chemical modification, improving heterogeneity derived from sugar chains, avoiding T cell epitopes that reduce immunogenicity identified by in silico prediction or by analysis using in vitro T cells, or introducing T cell epitopes that regulate T cell activation, etc. (mAbs 3: 243-247, 2011).

[0140] Whether the variable region of the antibody of the present invention "can bind to the first antigen and the second antigen" can be determined using a known method.

[0141] For example, it can be measured by electrochemiluminescence method (ECL method) (BMC Research Notes 2011, 4: 281).

[0142] Specifically, for example, the first antigen or the second antigen labeled with sulfo-tag (Ru complex) is mixed into a region where the tested antigen-binding molecule labeled with biotin can bind to the first antigen and the second antigen, such as a low-molecular antibody composed of a Fab region or a monovalent antibody (an antibody lacking one of the two Fab regions possessed by a common antibody), and the resulting mixture is added to a streptavidin-immobilized plate. At this time, the tested antigen-binding molecule labeled with biotin binds to the streptavidin on the plate. The Sulfo-tag is made luminescent, and by detecting its luminescent signal using Sector Imager 600, 2400 (MSD), etc., the binding of the first antigen or the second antigen to the above-mentioned region of the tested antigen-binding molecule can be confirmed.

[0143] Alternatively, the protein may be measured by ELISA, FACS (fluorescence activated cell sorting), ALPHA screening (Amplified Luminescent Proximity Homogeneous Assay), or the BIACORE method utilizing the surface plasmon resonance (SPR) phenomenon (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0144] Specifically, for example, Biacore (GE Healthcare) as an interaction analysis instrument utilizing the surface plasmon resonance (SPR) phenomenon can be used to measure. Biacore also includes any of Biacore T100, T200, X100, A100, 4000, 3000, 2000, 1000, C, etc. Any of the Biacore sensor chips of CM7, CM5, CM4, CM3, C1, SA, NTA, L1, HPA, Au chips, etc. can also be used on the sensor chip. The 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, antigen peptide, etc. that supplement the antigen binding molecules of the present invention are fixed with proteins by coupling methods such as amine coupling, disulfide coupling, aldehyde coupling, etc. on the sensor chip. Make the first antigen or the second antigen flow through the sensor chip as an analyte, measure the interaction, and obtain a sensorgram. The concentration of the first antigen or the second antigen at this time can be implemented in the range of several μM to several pM according to the strength of the interaction such as KD of the sample to be measured.

[0145] In addition, the first antigen or the second antigen may be immobilized on the sensor chip instead of the antigen-binding molecule, and the antibody sample to be evaluated may interact with the sensor chip. Based on the dissociation constant (KD) value calculated from the sensorgram of the interaction, or the degree of increase in the sensorgram before and after the action of the antigen-binding molecule sample, it can be determined whether the antibody variable region of the antigen-binding molecule of the present invention has binding activity to the first antigen or the second antigen.

[0146] ALPHA screening is implemented based on the following principle using the ALPHA technology using two types of beads, donor and acceptor. The molecules bound to the donor beads and the molecules bound to the acceptor beads undergo biological interactions, and a luminescent signal is detected only when the two beads are in close proximity. The photosensitizer in the donor beads excited by the laser converts the surrounding oxygen into singlet oxygen in an excited state. The singlet oxygen diffuses around the donor beads, and when it reaches the nearby acceptor beads, it causes a chemiluminescent reaction in the beads, ultimately emitting light. When the molecules bound to the donor beads and the molecules bound to the acceptor beads do not interact, the singlet oxygen generated by the donor beads will not reach the acceptor beads, and thus will not cause a chemiluminescent reaction.

[0147] One side (ligand) of the substance to be observed for interaction is fixed on the gold film of the sensor chip. If light is irradiated from the back side of the sensor chip so that total reflection occurs at the interface of the gold film and the glass, a part of the reflected light will form a part (SPR signal) with reduced reflection intensity. The other side (analyte) of the substance to be observed for interaction flows through the surface of the sensor chip. If the ligand and the analyte are combined, the mass of the immobilized ligand molecule will increase, and the refractive index of the solvent on the surface of the sensor chip will change. Due to the change in the refractive index, the position of the SPR signal shifts (on the contrary, if the combination dissociates, the position of the signal will return). The Biacore system uses the amount of the above-mentioned shift, i.e., the mass change on the surface of the sensor chip, as the vertical axis, and represents the time change of the mass as the measurement data (sensor diagram). The binding amount of the analyte and the ligand supplemented to the surface of the sensor chip can be obtained from the sensor diagram (the amount of change in the response on the sensor diagram before and after the analyte interacts). However, since the binding amount also depends on the amount of the ligand, when making a comparison, the amount of the ligand must be compared under the condition of substantially the same amount. In addition, the kinetic parameters: association rate constant (ka) and dissociation rate constant (kd) can be obtained from the sensorgram curve, and the affinity (KD) can be obtained from the ratio of the constants. Inhibition assays are also suitable for use in the BIACORE method. Examples of inhibition assays are described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

[0148] Whether the antigen-binding molecule of the present invention "does not bind to the first antigen and the second antigen at the same time" can be confirmed as follows: after confirming that it has binding activity to the first antigen and the second antigen, either the first antigen or the second antigen is allowed to bind to an antigen-binding molecule containing a variable region having such binding activity in advance, and then the above-mentioned method is used to determine whether it has binding activity to the remaining antigen.

[0149] Alternatively, the binding of an antigen-binding molecule to either the first antigen or the second antigen immobilized on an ELISA plate or a sensor chip can be confirmed by adding the other antigen to a solution and measuring whether the binding is inhibited.

[0150] Specifically, for example, when using the ECL method, a first antigen labeled with a sulfo-tag (Ru complex) and an unlabeled second antigen are prepared for the tested antigen-binding molecule labeled with biotin. When the tested antigen-binding molecule can bind to the first antigen and the second antigen, but not to the first antigen and the second antigen at the same time, in the absence of the unlabeled second antigen, the mixture of the tested antigen-binding molecule and the first antigen is added to a streptavidin immobilized plate, and if the sulfo-tag is made luminescent, its luminescent signal can be detected, but in the presence of the second antigen, the luminescent signal will decrease. The relative binding activity can be determined by quantitatively reducing the luminescent signal.

[0151] In addition, in the case of ALPHA screening, in the absence of the second antigen of competition, the tested antigen binding molecule interacts with the first antigen and produces a signal of 520-620nm. The second antigen without labeling competes with the tested antigen binding molecule for the interaction with the first antigen. By quantifying the reduction of the fluorescence reflecting the competition result, the relative binding activity can be determined. It is known that the polypeptide is biotinylated using Sulfo-NHS-biotin, etc. As a method for labeling the first antigen with GST, it is suitable to express the polynucleotide encoding the first antigen and the polynucleotide encoding GST in the cells of the vector of the fusion gene formed by the expression frame, and utilize the method of glutathione column purification, etc. The obtained signal can be suitably analyzed by using software such as GRAPHPAD PRISM (GraphPad Society, San Diego), and the one-site competition (one-site competition) model fitting utilizing nonlinear regression analysis. At this time, the second antigen is labeled, and the first antigen is not labeled and can also be analyzed in the same manner.

[0152] In addition, the method utilizing fluorescence resonance energy transfer (FRET, Fluorescence Resonance Energy Transfer) can also be used. FRET refers to the phenomenon that the excitation energy is directly transferred by electronic resonance between two fluorescent molecules close to each other. If FRET is caused, the excitation energy of the donor (fluorescent molecule in an excited state) is transferred to the acceptor (another fluorescent molecule near the donor), so the fluorescence emitted from the donor disappears (to be precise, the fluorescence lifetime is shortened), and on the contrary, fluorescence is emitted from the acceptor. This phenomenon can be used to analyze whether it is dual-Fab. For example, if the second antigen with a fluorescent donor first antigen and a fluorescent acceptor is introduced and combined with the tested antigen binding molecule at the same time, the fluorescence of the donor disappears, and fluorescence is emitted from the acceptor, thus causing a change in fluorescence wavelength. Such an antibody can be judged as non-dual-Fab. On the other hand, when the first antigen, the second antigen, and the tested antigen binding molecule are mixed, if the change in the fluorescence wavelength of the fluorescent donor combined with the first antigen is not caused, the tested antigen binding molecule can be considered to be dual-Fab.

[0153] In addition, for example, on donor beads, the tested antigen binding molecules through biotin labeling are combined with the streptavidin on the donor beads, and the first antigen through glutathione S transferase (GST) labeling is combined with the acceptor beads. In the absence of the second antigen of competition, the tested antigen binding molecules interact with the first antigen and produce a signal of 520-620nm. The second antigen without labeling competes with the tested antigen binding molecules for the interaction with the first antigen. By quantitatively reducing the fluorescence reflecting the competition result, relative binding activity can be determined. It is known that polypeptides are biotinylated using Sulfo-NHS-biotin, etc. As a method for the first antigen labeling with GST, it can be suitably adopted to express the polynucleotides encoding the first antigen and the polynucleotides encoding GST in the cells etc. of the vectors of the fusion gene formed by fusion in the expression frame, and utilize the method of glutathione column purification, etc. The obtained signal can be suitably analyzed by fitting to a one-site competition model by nonlinear regression analysis using software such as GraphPad PRISM (GraphPad, San Diego).

[0154] It should be noted that labeling is not limited to GST, and can be labeling with any label such as histidine tag, MBP, CBP, Flag tag, HA tag, V5 tag, c-myc tag, etc., without limitation. In addition, about the combination of the tested antigen binding molecule and the donor beads, it is not limited to the combination using biotin-streptavidin reaction. In particular, when the tested antigen binding molecule contains Fc, it can be considered that the Fc recognition protein of protein A, protein G, etc. on the donor beads makes the tested antigen binding molecule bind.

[0155] In addition, the situation in which the first antigen and the second antigen can bind to both the first antigen and the second antigen simultaneously when they are not expressed on the cell membrane like soluble proteins, or when they are present on the same cell, but cannot bind to both when they are expressed on different cells, can also be measured by using a known method.

[0156] Specifically, even if it is positive in the ECL-ELISA that detects simultaneous binding with the first antigen and the second antigen, when the cells expressing the first antigen, the cells expressing the second antigen and the tested antigen binding molecules are mixed, if these three do not bind at the same time, when expressed on different cells, it can be shown that they do not bind at the same time. For example, it can be measured by the ECL-ELISA method using cells. The cells expressing the first antigen are fixed to the plate in advance, and after the tested antigen binding molecules are bound, the cells expressing the second antigen are added. By detecting the other antigens that are only expressed on the cells expressing the second antigen, using sulfo-tag labeled antibodies, when the two antigens expressed on the two cells are combined at the same time, a signal is observed, and when they are not combined at the same time, no signal is observed.

[0157] Alternatively, the assay can be performed by the alpha screen method. When cells expressing the first antigen bound to donor beads, cells expressing the second antigen bound to acceptor beads and the antigen-binding molecule to be tested are mixed, a signal can be observed when the two antigens expressed on the two cells are simultaneously bound, but no signal can be observed when the two antigens are not simultaneously bound.

[0158] Alternatively, the interaction analysis method using Octet can be used for determination. First, initially, cells expressing the first antigen with a peptide tag are combined with a biosensor that recognizes the peptide tag. When interaction analysis is performed in a hole in which cells expressing the second antigen and the tested antigen binding molecule have been added, when the two antigens expressed on the two cells are combined simultaneously, the tested antigen binding molecule and the cells expressing the second antigen are combined with the biosensor, thereby observing a larger wavelength shift, and when not combined simultaneously, only the tested antigen binding molecule is combined with the biosensor, thereby observing a smaller wavelength shift.

[0159] Alternatively, in addition to binding activity, the determination of biological activity can also be carried out. For example, when the cell expressing the first antigen, the cell expressing the second antigen and the tested antigen binding molecule are mixed and cultured, when the two antigens expressed on the two cells are combined simultaneously, the activation is mutually activated via the tested antigen binding molecule, so the changes of activation signals such as the increase of phosphorylation in the downstream of the respective antigens can be detected. Alternatively, as a result of activation, the generation of cytokines is induced, so by measuring the production amount of cytokines, it can also be judged whether to combine with the two cells simultaneously.

[0160] In the present invention, "Fc region" refers to a region comprising a fragment consisting of a hinge portion or a part thereof, a CH2, and a CH3 domain in an antibody molecule. The Fc region of the IgG class refers to a region represented by EU numbering (also referred to as EU INDEX in this specification), such as cysteine ​​at position 226 to the C-terminus, or proline at position 230 to the C-terminus, but is not limited thereto. The Fc region can be appropriately obtained by the following method: IgG1, IgG2, IgG3, IgG4 monoclonal antibodies, etc. are partially digested with a proteolytic enzyme such as pepsin, adsorbed on a protein A column or a protein G column, and then the adsorbed components are eluted to obtain. As the proteolytic enzyme, by appropriately setting the reaction conditions of the enzyme such as pH, as long as the full-length antibody can be digested to restrictively produce Fab or F(ab')2, there is no particular limitation, for example, pepsin or papain, etc.

[0161] The "antigen binding molecule" in the present invention is not particularly limited as long as it is a molecule comprising the "antibody variable region" of the present invention, and further, it may include a peptide or protein having a length of about 5 amino acids or more. It is not limited to a peptide or protein of biological origin, for example, it may be a polypeptide consisting of an artificially designed sequence. Moreover, it may be any of a natural polypeptide, a synthetic polypeptide, a recombinant polypeptide, etc.

[0162] Preferred examples of the antigen-binding molecules of the present invention include antigen-binding molecules comprising an antibody Fc region.

[0163] As the "Fc region" of the present invention, for example, an Fc region derived from natural IgG can be used. Here, natural IgG refers to a polypeptide that contains the same amino acid sequence as IgG found in nature and belongs to the antibody class substantially encoded by the immunoglobulin γ gene. For example, natural human IgG refers to natural human IgG1, natural human IgG2, natural human IgG3, natural human IgG4, etc. Natural IgG also includes mutants naturally produced therefrom. As the constant region of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, multiple allotypic sequences caused by genetic polymorphism are recorded in Sequences of proteins of immunological interest, NIH Publication No.91-3242, and the present invention can be any of the sequences therein. In particular, as the sequence of human IgG1, the amino acid sequence at EU numbering 356-358 can be DEL or EEM.

[0164] As the Fc region of the antibody, for example, there are Fc regions of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM categories. The Fc region of the antibody of the present invention, for example, can use the Fc region derived from a natural human IgG antibody. As the Fc region of the present invention, for example, the Fc region derived from the constant region of a natural IgG can be used, specifically, the constant region (SEQ ID NO: 1) derived from natural human IgG1, the constant region (SEQ ID NO: 2) derived from natural human IgG2, the constant region (SEQ ID NO: 3) derived from natural human IgG3, and the constant region (SEQ ID NO: 4) derived from natural human IgG4 can be used. The constant region of natural IgG also includes naturally occurring mutants, etc.

[0165] As the Fc region of the present invention, an Fc region with reduced binding activity to Fcγ receptors is particularly preferred. Here, Fcγ receptor (sometimes described as Fcγ receptor, FcγR or Fcγ receptor in this specification) refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, and IgG4, and substantially also means any member of the protein family encoded by the Fcγ receptor gene. For humans, the family also includes: FcγRI (CD64) containing isotypes FcγRIa, FcγRIb and FcγRIc; FcγRII (CD32) containing isotypes FcγRIIa (containing allotypes H131 (H type) and R131 (R type)), FcγRIIb (containing FcγRIIb-1 and FcγRIIb-2) and FcγRIIc; and FcγRIII (CD16) containing isotypes FcγRIIIa (containing allotypes V158 and F158) and FcγRIIIb (containing allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2); and any undiscovered human FcγR class or FcγR isotype or allotype, but is not limited to this. FcγR includes FcγR derived from humans, mice, rats, rabbits and monkeys, but is not limited thereto and may be derived from any organism. Mouse FcγRs also include: FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16) and FcγRIII-2 (CD16-2) and any undiscovered mouse FcγRs or FcγR isoforms or allotypes, but are not limited thereto. Preferred examples of such Fcγ receptors include: human FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16) and / or FcγRIIIb (CD16).

[0166] FcγRs include activating receptors with ITAM (Immunoreceptor tyrosine-based activation motif) and inhibitory receptors with ITIM (immunoreceptor tyrosine-based inhibitory motif). FcγRs are classified into activating FcγRs of FcγRI, FcγRIIa R, FcγRIIa H, FcγRIIIa, and FcγRIIIb, and inhibitory FcγRs of FcγRIIb.

[0167] The polynucleotide sequence and amino acid sequence of FcγRI are recorded in NM_000566.3 and NP_000557.1, respectively; the polynucleotide sequence and amino acid sequence of FcγRIIa are recorded in BC020823.1 and AAH20823.1, respectively; the polynucleotide sequence and amino acid sequence of FcγRIIb are recorded in BC146678.1 and AAI46679.1, respectively; the polynucleotide sequence and amino acid sequence of FcγRIIIa are recorded in BC033678.1 and AAH33678.1, respectively; and the polynucleotide sequence and amino acid sequence of FcγRIIIb are recorded in BC128562.1 and AAI28563.1, respectively (RefSeq accession numbers). In addition, there are two gene polymorphisms in FcγRIIa, where the amino acid at position 131 of FcγRIIa is substituted with histidine (H type) or arginine (R type) (J. Exp. Med, 172, 19-25, 1990). In addition, there are two gene polymorphisms in FcγRIIb, where the amino acid at position 232 of FcγRIIb is substituted with isoleucine (I type) or threonine (T type) (Arthritis. Rheum. 46: 1242-1254 (2002)). In addition, there are two gene polymorphisms in FcγRIIIa, where the amino acid at position 158 of FcγRIIIa is substituted with valine (V type) or phenylalanine (F type) (J. Clin. Invest. 100 (5): 1059-1070 (1997)). Furthermore, there are two types of genetic polymorphisms in FcγRIIIb, NA1 type and NA2 type (J. Clin. Invest. 85: 1287-1295 (1990)).

[0168] Whether the binding activity to Fcγ receptors is reduced can be confirmed by well-known methods such as FACS, ELISA format, ALPHA screening (Amplified Luminescent Proximity Homogeneous Assay), or BIACORE method utilizing surface plasmon resonance (SPR) phenomenon (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0169] ALPHA screening is implemented based on the following principle using the ALPHA technology using two types of beads, donor and acceptor. The molecules bound to the donor beads and the molecules bound to the acceptor beads undergo biological interactions, and a luminescent signal is detected only when the two beads are in close proximity. The photosensitizer in the donor beads excited by the laser converts the surrounding oxygen into singlet oxygen in an excited state. The singlet oxygen diffuses around the donor beads, and when it reaches the nearby acceptor beads, it causes a chemiluminescent reaction in the beads, ultimately emitting light. When the molecules bound to the donor beads and the molecules bound to the acceptor beads do not interact, the singlet oxygen generated by the donor beads will not reach the acceptor beads, and thus will not cause a chemiluminescent reaction.

[0170] For example, the antigen binding molecules through biotin labeling are combined with donor beads, and the Fcγ receptors through glutathione S transferase (GST) labeling are combined with acceptor beads. In the absence of antigen binding molecules with competitive variant Fc regions, antigen binding molecules with native Fc regions interact with Fcγ receptors and produce a signal of 520-620nm. Antigen binding molecules with unlabeled variant Fc regions compete with antigen binding molecules with native Fc regions for interaction with Fcγ receptors. By quantifying the reduction of the fluorescence reflecting the competition result, relative binding affinity can be determined. It is known to use Sulfo-NHS-biotin etc. to biotinylate antigen binding molecules such as antibodies. As a method for labeling Fcγ receptors with GST, it can be suitably adopted to express the polynucleotides encoding Fcγ receptors and the polynucleotides encoding GST in the cells etc. of the vectors of the fusion gene formed by fusion in the expression frame, and utilize the method of glutathione column purification etc. The obtained signal can be suitably analyzed by fitting to a one-site competition model using nonlinear regression analysis using software such as GraphPad PRISM (GraphPad, San Diego).

[0171] One side of the substance to be observed for interaction (ligand) is fixed on the gold film of the sensor chip. If light is irradiated from the back side of the sensor chip so that total reflection occurs at the interface between the gold film and the glass, a part of the reflected light will form a part with reduced reflection intensity (SPR signal). The other side of the substance to be observed for interaction (analyte) flows over the surface of the sensor chip. If the ligand and the analyte are combined, the mass of the immobilized ligand molecule will increase, and the refractive index of the solvent on the surface of the sensor chip will change. Due to the change in the refractive index, the position of the SPR signal shifts (on the contrary, if the combination dissociates, the position of the signal will return). The Biacore system uses the amount of the above-mentioned shift, that is, the mass change on the surface of the sensor chip, as the vertical axis, and the time change of the mass as the measurement data (sensor graph). The kinetic parameters can be obtained from the curve of the sensor graph: the binding rate constant (ka) and the dissociation rate constant (kd), and the affinity (KD) can be obtained from the ratio of the constants. The inhibition assay is also suitable for use in the BIACORE method. Examples of inhibition assays are described in Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010.

[0172] As used herein, "reduced Fcγ receptor binding activity" means, for example, that the binding activity of the test antigen-binding molecule shows 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 Fc region-containing antigen-binding molecule according to the above-mentioned analysis method.

[0173] As a control antigen-binding molecule, an antigen-binding molecule having an Fc region of an IgG1, IgG2, IgG3 or IgG4 monoclonal antibody can be suitably used. The structure of the Fc region is described in: SEQ ID NO: 1 (A is added to the end of RefSeq accession number AAC82527.1), SEQ ID NO: 2 (A is added to the end of RefSeq accession number AAB59393.1), SEQ ID NO: 3 (A is added to the end of RefSeq accession number CAA27268.1), SEQ ID NO: 4 (A is added to the end of RefSeq accession number AAB59394.1). In addition, when an antigen-binding molecule having an Fc region mutant of an antibody of a certain isotype is used as a test substance, the effect of the binding activity to Fcγ receptors caused by the mutation of the mutant can be verified by using an antigen-binding molecule having an Fc region of an antibody of the specific isotype as a control. As described above, an antigen-binding molecule having a mutant of an Fc region whose binding activity to Fcγ receptors is reduced and verified is suitably prepared.

[0174] As examples of such mutants, mutants such as deletion of 231A-238S of amino acids specified by EU numbering (WO2009 / 011941), C226S, C229S, P238S, (C220S) (J. Rheumatol (2007) 34, 11), C226S, C229S (Hum. Antibod. Hybridomas (1990) 1 (1), 47-54), C226S, C229S, E233P, L234V, L235A (Blood (2007) 109, 1185-1192) are well known.

[0175] That is, preferred examples include an antigen-binding molecule having an Fc region in which any one of the following amino acids specified by EU numbering: 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, or 332, among the amino acids constituting the Fc region of an antibody of a specific isotype, is substituted. The antibody isotype from which the Fc region is derived is not particularly limited, and an Fc region derived from an IgG1, IgG2, IgG3 or IgG4 monoclonal antibody can be appropriately used, and an Fc region derived from a natural human IgG1 antibody is preferably used.

[0176] For example, an antigen-binding molecule having an Fc region in which any of the following groups of substitutions specified according to EU numbering are made in the amino acids constituting the Fc region of an IgG1 antibody (the numbers indicate the positions of the amino acid residues specified according to EU numbering, the single-letter amino acid symbols before the numbers indicate the amino acid residues before substitution, and the single-letter amino acid symbols after the numbers indicate the amino acid residues before substitution), or an Fc region in which the amino acid sequence at positions 231 to 238 is deleted can also be suitably used.

[0177] (a) L234F, L235E, P331S;

[0178] (b) C226S, C229S, P238S;

[0179] (c) C226S, C229S;

[0180] (d)C226S, C229S, E233P, L234V, L235A.

[0181] In addition, antigen-binding molecules having an Fc region in which any of the following groups of substitutions specified by EU numbering are performed in the amino acids constituting the Fc region of an IgG2 antibody (the numbers indicate the positions of amino acid residues specified by EU numbering, the single-letter amino acid symbol before the numbers indicates the amino acid residue before substitution, and the single-letter amino acid symbol after the numbers indicates the amino acid residue before substitution) can also be suitably used:

[0182] (e)H268Q, V309L, A330S, P331S;

[0183] (f) V234A;

[0184] (g) G237A;

[0185] (h) V234A, G237A;

[0186] (i) A235E, G237A;

[0187] (j) V234A, A235E, G237A.

[0188] In addition, antigen-binding molecules having an Fc region in which any of the following groups of substitutions specified by EU numbering are performed in the amino acids constituting the Fc region of an IgG3 antibody (the numbers indicate the positions of amino acid residues specified by EU numbering, the single-letter amino acid symbol before the numbers indicates the amino acid residue before substitution, and the single-letter amino acid symbol after the numbers indicates the amino acid residue before substitution) can also be suitably used:

[0189] (k) F241A;

[0190] (l) D265A;

[0191] (m)V264A.

[0192] In addition, antigen-binding molecules having an Fc region in which any of the following groups of substitutions specified by EU numbering are performed in the amino acids constituting the Fc region of an IgG4 antibody (the numbers indicate the positions of amino acid residues specified by EU numbering, the single-letter amino acid symbol before the numbers indicates the amino acid residue before substitution, and the single-letter amino acid symbol after the numbers indicates the amino acid residue before substitution) can also be suitably used:

[0193] (n) L235A, G237A, E318A;

[0194] (o) L235E;

[0195] (p)F234A, L235A.

[0196] As another preferred example, an antigen-binding molecule having an Fc region can be cited, wherein the Fc region is an Fc region in which any one of the following amino acids specified by EU numbering: 233, 234, 235, 236, 237, 327, 330, 331, among the amino acids constituting the Fc region of a native human IgG1 antibody, is substituted with an amino acid corresponding to its EU numbering in the corresponding IgG2 or IgG4.

[0197] As another preferred example, an antigen-binding molecule having an Fc region in which any one or more of the following amino acids specified by EU numbering: 234, 235, and 297, among the amino acids constituting the Fc region of a native human IgG1 antibody, are substituted with other amino acids can be preferably mentioned. The type of amino acids present after the substitution is not particularly limited, and an antigen-binding molecule having an Fc region in which any one or more of the amino acids at positions 234, 235, and 297 are substituted with alanine is particularly preferred.

[0198] As another preferred example, an antigen-binding molecule having an Fc region in which any one of the following amino acids specified by EU numbering is substituted with another amino acid at position 265 among the amino acids constituting the Fc region of an IgG1 antibody can be preferably mentioned. The type of amino acid present after the substitution is not particularly limited, and an antigen-binding molecule having an Fc region in which the amino acid at position 265 is substituted with alanine is particularly preferred.

[0199] Furthermore, one preferred embodiment of the "antigen-binding molecule" of the present invention includes a multispecific antibody comprising the variable regions of the antibodies of the present invention.

[0200] In the association of multispecific antibodies, the following technology can be applied: a technology that introduces charge repulsion into the interface of the second constant region (CH2) or the third constant region (CH3) of the antibody H chain to suppress the association of non-target H chains (WO2006 / 106905).

[0201] In the technique of introducing charge repulsion into the interface of CH2 or CH3 to suppress the undesired association between H chains, examples of amino acid residues that contact at the interface of other constant regions of the H chain include residues at EU numbering position 356, residues at EU numbering position 439, residues at EU numbering position 357, residues at EU numbering position 370, residues at EU numbering position 399, and residues at EU numbering position 409 in the CH3 region.

[0202] More specifically, for example, in an antibody comprising two H chain CH3 regions, an antibody can be formed in which one to three groups of amino acid residues selected from the following amino acid residue groups (1) to (3) in the first H chain CH3 region have the same charge:

[0203] (1) the amino acid residues contained in the H chain CH3 region and the amino acid residues at positions 356 and 439 in EU numbering;

[0204] (2) the amino acid residues contained in the H chain CH3 region and the amino acid residues at positions 357 and 370 by EU numbering;

[0205] (3) The amino acid residues contained in the H chain CH3 region and the amino acid residues at positions 399 and 409 in EU numbering.

[0206] Furthermore, the following antibodies can be formed: an antibody in which one to three groups of amino acid residues corresponding to the groups of amino acid residues shown in (1) to (3) in the second H chain CH3 region different from the groups of amino acid residues shown in (1) to (3) in the first H chain CH3 region have the same charge as that in the first H chain CH3 region, and the corresponding amino acid residues in the first H chain CH3 region have a charge opposite to that in the first H chain CH3 region.

[0207] The amino acid residues described in (1) to (3) above are close to each other when associated. Regarding the desired H chain CH3 region or H chain constant region, those skilled in the art can find the sites corresponding to the amino acid residues described in (1) to (3) above by performing homology modeling using commercially available software, and can appropriately modify the amino acid residues at the sites.

[0208] In the above-mentioned antibody, the “charged amino acid residue” is, for example, preferably an amino acid residue selected from the group consisting of (a) or (b) below:

[0209] (a) Glutamic acid (E), aspartic acid (D);

[0210] (b) Lysine (K), arginine (R), and histidine (H).

[0211] In the above-mentioned antibody, "having the same charge" means, for example, that two or more amino acid residues have amino acid residues included in any one of the above-mentioned groups (a) or (b). "Having opposite charges" means, for example, that when at least one of the two or more amino acid residues has an amino acid residue included in any one of the above-mentioned groups (a) or (b), the remaining amino acid residues have amino acid residues included in a different group.

[0212] In a preferred embodiment, the first H chain CH3 region and the second H chain CH3 region of the above-mentioned antibody may be cross-linked via a disulfide bond.

[0213] In the present invention, the amino acid residues to be modified are not limited to the above-mentioned amino acid residues in the variable region or constant region of the antibody. Regarding polypeptide mutants or heteromultimers, those skilled in the art can find the amino acid residues forming the interface by performing homology modeling using commercially available software, and can modify the amino acid residues at the site to control the association.

[0214] In addition, other known techniques can be applied to the association of the multispecific antibodies of the present invention. Substituting the amino acid side chains present in the variable region of one H chain of the antibody with larger side chains (knobs) and substituting the amino acid side chains present in the opposite variable region of the other H chain with smaller side chains (holes) so that the knobs can be arranged in the holes can effectively cause the association of polypeptides containing different amino acids in the Fc region (WO1996 / 027011, Ridgway JB et al., Protein Engineering (1996) 9, 617-621, Merchant AM et al. Nature Biotechnology (1998) 16, 677-681).

[0215] In addition, in the formation of the multispecific antibody of the present invention, other known techniques can also be applied. By using a strand-exchange engineered domain CH3 in which a portion of CH3 of one H chain of an antibody is used as an IgA-derived sequence corresponding to the portion, and a sequence corresponding to the IgA-derived sequence of the portion is introduced into the complementary portion of CH3 of the other H chain, the association of polypeptides with different sequences can be effectively caused by the complementary association of CH3 (Protein Engineering Design & Selection, 23; 195-202, 2010). Even using this known technique, the target multispecific antibody can be effectively formed.

[0216] In addition, in the formation of multispecific antibodies, the following technologies can also be used: the antibody production technology using the CH1-CL association and VH-VL association of antibodies described in WO2011 / 028952; the technology for producing bispecific antibodies using separately modulated monoclonal antibody partners (Fab ArmExchange) described in WO2008 / 119353 or WO2011 / 131746; the technology for controlling the association between CH3 of antibody heavy chains described in WO2012 / 058768 or WO2013 / 063702; the technology for producing bispecific antibodies composed of two light chains and one heavy chain described in WO2012 / 023053; Christoph et al. (Nature Biotechnology Vol.31, p753-758 (2013)) described in the technique of producing bispecific antibodies using two bacterial cell lines that express a half-molecule of an antibody composed of one H chain and one L chain, respectively. In addition to the above-mentioned association technique, CrossMab technology (Scaefer et al. (Proc. Natl. Acad. Sci. USA (2011) 108, 11187-11192)), which is known as an association technique for making a light chain that forms a variable region that binds to a first epitope and a light chain that forms a variable region that binds to a second epitope associate with a heterogeneous light chain that forms a heavy chain that forms a variable region that binds to a first epitope and a heavy chain that forms 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 preparing bispecific antibodies using separately prepared monoclonal antibody partners, there can be mentioned a method of obtaining a desired bispecific antibody by placing a monoclonal antibody in which a specific amino acid present in the heavy chain CH3 region is substituted under reducing conditions to promote the heterologous nature of the antibody. Preferred amino acid substitution sites in this method include, for example, residues at EU numbering 392 and residues at EU numbering 397 in the CH3 region. Furthermore, a dual-specific antibody can be prepared using an antibody in which one to three groups of amino acid residues selected from the following amino acid residue groups (1) to (3) in the CH3 region of the first H chain have the same charge: (1) amino acid residues contained in the CH3 region of the H chain and amino acid residues at EU numbering 356 and 439; (2) amino acid residues contained in the CH3 region of the H chain and amino acid residues at EU numbering 357 and 370; (3) amino acid residues contained in the CH3 region of the H chain and amino acid residues at EU numbering 399 and 409.Furthermore, a dual-specific antibody can be prepared using one to three groups of amino acid residues corresponding to the groups of amino acid residues shown in (1) to (3) in the CH3 region of the second H chain that are different from the groups of amino acid residues shown in (1) to (3) in the CH3 region of the first H chain, and having the same charge as that of the corresponding amino acid residues in the CH3 region of the first H chain, wherein the amino acid residues have the opposite charge to those in the CH3 region of the first H chain.

[0217] In addition, even in the case where the target multispecific antibody cannot be effectively formed, the multispecific antibody of the present invention can be obtained by separating and purifying the target multispecific antibody from the generated antibody. For example, it is reported that the difference in isoelectric point can be given by introducing amino acid substitutions into the variable regions of the two H chains, and the two homodimers and the target heterodimer antibodies can be purified by ion exchange column chromatography (WO2007114325). In addition, as a method for purifying heterodimers, it has been reported so far that the heterodimerization antibody composed of the H chain of mouse IgG2a bound to protein A and the H chain of rat IgG2b not bound to protein A is purified using protein A (WO98050431, WO95033844). Furthermore, by using the amino acid residues at positions 435 and 436 of the EU numbering as the binding site of IgG and protein A to replace the amino acid residues at positions 435 and 436 of the EU numbering as Tyr, His, etc., which have different binding abilities with protein A, the interaction between each H chain and protein A is changed, and by using a protein A column, only the heterodimerization antibody can be effectively purified.

[0218] It is also possible to use a plurality of these techniques, for example, two or more in combination. In addition, these techniques can also be appropriately applied to the two H chains expected to associate. It should be noted that the antigen binding molecules of the present invention can also be prepared separately with the same amino acid sequence based on the addition of the above-mentioned changes.

[0219] The amino acid sequence can be modified by various methods known in the art. These methods are not limited to the following methods, but can be modified by the following methods: site-specific mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. Gene 152, 271-275; Zoller, MJ, and Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 100, 468-500; Kramer, W, Drutsa, V, Jansen, HW, Kramer, B, Pflugfelder, M, and Fritz, HJ (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456; Kramer W, and Fritz HJ (1987) Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367; Kunkel, TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci US A. 82, 488-492), PCR mutagenesis, cassette mutagenesis, etc.

[0220] In addition, the "antigen-binding molecule" of the present invention may be an antibody fragment that contains both the heavy chain and the light chain that form the "antibody variable region" of the present invention in a single polypeptide chain, but lacks the constant region. Such an antibody fragment may be, for example, a double-chain antibody (diabody; Db), a single-chain antibody, or sc(Fab')2.

[0221] Db is a dimer composed of two polypeptide chains (Holliger P et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); EP 404,097; W093 / 11161, etc.), and each polypeptide chain is connected by a relatively short linker (the L chain variable region (VL) and the H chain variable region (VH) cannot bind to each other in the same chain), for example, about 5 residues.

[0222] VL and VH encoded on the same polypeptide chain cannot form a single-chain variable region fragment due to the short linker between them, and thus form two antigen-binding sites by dimerization.

[0223] As a single-chain antibody, for example, sc(Fv)2 can be cited. sc(Fv)2 is a single-chain antibody in which four variable regions of two VLs and two VHs are connected by a linker such as a peptide linker to form a single chain (J Immunol. Methods (1999) 231 (1-2), 177-189). The two VHs and VLs can also be derived from different monoclonal antibodies. For example, it is also preferable to cite: bispecific (bispecific sc(Fv)2) that recognizes two epitopes present in the same antigen as disclosed in Journal of Immunology (1994) 152 (11), 5368-5374. sc(Fv)2 can be prepared by a method known to those skilled in the art. For example, it can be prepared by connecting scFv with a linker such as a peptide linker.

[0224] As the composition of the antigen-binding domain constituting sc(Fv)2 in this specification, an antibody characterized by the following sequence arrangement can be cited: two VHs and two VLs are arranged in the order of VH, VL, VH, VL ([VH] linker [VL] linker [VH] linker [VL]) based on the N-terminal side of the single-chain polypeptide, and the order of the two VHs and two VLs is not particularly limited to the above-mentioned composition, and can be arranged in any order. For example, a composition in the following order can also be cited.

[0225] [VL] Connector [VH] Connector [VH] Connector [VL]

[0226] [VH]Connector [VL]Connector [VL]Connector [VH]

[0227] [VH]Connector [VH]Connector [VL]Connector [VL]

[0228] [VL] Connector [VL] Connector [VH] Connector [VH]

[0229] [VL] Connector [VH] Connector [VL] Connector [VH]

[0230] The molecular format of sc(Fv)2 is also described in detail in WO2006 / 132352, and those skilled in the art can appropriately prepare the desired sc(Fv)2 based on this description to prepare the antigen-binding molecules disclosed in this specification.

[0231] Furthermore, the antigen-binding molecules of the present invention may be conjugated to carrier polymers such as PEG or organic compounds such as anticancer agents. In addition, a sugar chain adding sequence is inserted, and sugar chains are preferably added for the purpose of obtaining a desired effect.

[0232] As a linker for binding to the variable region of an antibody, any peptide linker or synthetic compound linker that can be introduced by genetic engineering (for example, refer to Protein Engineering, 9 (3), 299-305, 1996) disclosed in the invention can be used, and a peptide linker is preferred in the present invention. The length of the peptide linker is not particularly limited, and those skilled in the art can select it appropriately according to the purpose. The preferred length is 5 amino acids or more (the upper limit is not particularly limited, usually 30 amino acids or less, preferably 20 amino acids or less), and 15 amino acids are particularly preferred. When sc (Fv) 2 contains 3 peptide linkers, all peptide linkers of the same length can be used, or peptide linkers of different lengths can be used.

[0233] For example, in the case of a peptide linker, preferably:

[0234] Ser Gly·Ser

[0235] Gly·Gly·Ser

[0236] Ser·Gly·Gly

[0237] Gly·Gly·Gly·Ser(SEQ ID NO:5)

[0238] Ser·Gly·Gly·Gly (SEQ ID NO:6)

[0239] Gly·Gly·Gly·Gly·Ser(SEQ ID NO:7)

[0240] Ser·Gly·Gly·Gly·Gly (SEQ ID NO:8)

[0241] Gly·Gly·Gly·Gly·Gly·Ser(SEQ ID NO:9)

[0242] Ser·Gly·Gly·Gly·Gly·Gly (SEQ ID NO:10)

[0243] Gly·Gly·Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 11)

[0244] Ser·Gly·Gly·Gly·Gly·Gly·Gly (SEQ ID NO: 12)

[0245] (Gly·Gly·Gly·Gly·Ser(SEQ ID NO:7))n

[0246] (Ser·Gly·Gly·Gly·Gly(SEQ ID NO:8))n

[0247] [n is an integer greater than or equal to 1], etc. However, those skilled in the art can appropriately select the length or sequence of the peptide linker according to the purpose.

[0248] Synthetic chemical linkers (chemical cross-linkers) are cross-linkers commonly used in the cross-linking of peptides, such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidyloxycarbonyloxy)ethyl]sulfone (BSOCOES), bis[2-(sulfosuccinimidyloxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES), etc. These cross-linkers are commercially available.

[0249] When four antibody variable regions are combined, three linkers are usually required, and the same linker may be used for all of them, or different linkers may be used.

[0250] F(ab')2 comprises two light chains and two heavy chains, and the heavy chain comprises a constant region (a part of the CH1 domain and the CH2 domain) so that a disulfide bond between the chains is formed between the two heavy chains. The F(ab')2 constituting the polypeptide complex disclosed in the present specification can be suitably obtained by the following method: a full-length monoclonal antibody having a desired antigen-binding domain is partially digested with a proteolytic enzyme such as pepsin, and then the Fc fragment is adsorbed on a protein A column to be removed. As the proteolytic enzyme, the enzyme reaction conditions such as pH are appropriately set, as long as the full-length antibody can be digested to restrictively produce F(ab')2, there is no particular limitation, and examples thereof include pepsin or ficin.

[0251] Furthermore, the antigen-binding molecules of the present invention may contain additional changes in addition to the above-mentioned amino acid changes. Additional changes may be selected from, for example, amino acid substitution, deletion, or modification, or a combination thereof.

[0252] For example, in the antigen binding molecules of the present invention, in the scope that the target function of this molecule does not produce substantial changes, can also arbitrarily add change.For example, such variation can be carried out by the conservative substitution of amino-acid residue.In addition, even if the target function of antigen binding molecules of the present invention produces the change of change, as long as the variation of this function can also carry out such change within the scope of target of the present invention.

[0253] The changes in the amino acid sequence of the present invention also include post-translational modifications. As a specific post-translational modification, the addition or deletion of sugar chains can be shown. For example, when the antigen-binding molecule of the present invention has a constant region of IgG1 type, the amino acid residue at position 297 of EU numbering can be an amino acid residue modified by sugar chains. There is no limitation on the modified sugar chain structure. Usually, antibodies expressed in eukaryotic cells have constant regions that contain sugar chain modifications. Therefore, antibodies expressed in the following cells can usually be modified by certain sugar chains.

[0254] Mammalian antibody-producing cells

[0255] Eukaryotic cells transformed with an expression vector containing antibody-encoding DNA

[0256] The eukaryotic cells shown here include yeast or animal cells. For example, CHO cells or HEK293H cells are representative animal cells for transformation with expression vectors containing DNA encoding antibodies. On the other hand, antibodies without sugar chain modification at this site are also included in the antibodies of the present invention. Antibodies without sugar chain modification in the constant region can be obtained by expressing the gene encoding the antibody in prokaryotic cells such as Escherichia coli.

[0257] In the present invention, the additional modification may be, more specifically, a modification in which sialic acid is added to the sugar chain of the Fc region (MAbs. 2010 Sep-Oct; 2(5): 519-27).

[0258] Furthermore, when the antigen-binding molecules of the present invention have an Fc region, for example, amino acid substitutions that improve FcRn-binding activity (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 amino acid substitutions that improve antibody heterogeneity or stability ((WO / 2009 / 041613)) can be added.

[0259] Furthermore, the term "antibody" in the present invention is used in the broadest sense, and includes any antibody such as monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody mutants, antibody fragments, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, and humanized antibodies, as long as they exhibit the desired biological activity.

[0260] The antibody of the present invention is not limited to the type of antigen, the origin of the antibody, etc., and may be any antibody. The origin of the antibody is not particularly limited, and examples thereof include human antibodies, mouse antibodies, rat antibodies, rabbit antibodies, and the like.

[0261] Methods for making antibodies are well known to those skilled in the art. For example, in the case of monoclonal antibodies, they can be prepared by the hybridoma method (Kohler and Milstein, Nature 256: 495 (1975)) and the recombinant method (U.S. Patent No. 4,816,567). In addition, they can be isolated from phage antibody libraries (Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1991)). In addition, they can be isolated from a single B cell clone (N. Biotechnol. 28 (5): 253-457 (2011)).

[0262] Humanized antibodies are also referred to as reshaped human antibodies. Specifically, humanized antibodies, etc., in which the CDR of an animal other than a human, such as a mouse antibody, is transplanted into a human antibody are well known. The usual gene recombination method for obtaining humanized antibodies is also known. Specifically, as a method for transplanting the CDR of a mouse antibody into a human FR, for example, overlap extension PCR is well known.

[0263] The DNA encoding the antibody variable region obtained by connecting 3 CDRs and 4 FRs and the DNA encoding the human antibody constant region are inserted into the expression vector in a frame fusion mode, thereby making a humanized antibody expression vector. After the integration vector (recombinant vector) is introduced into the host to establish a recombinant cell, the recombinant cell is cultured, and the humanized antibody is produced in the culture of the cultured cell by expressing the DNA encoding the humanized antibody (refer to European Patent Publication EP239400, International Publication WO1996 / 002576).

[0264] If necessary, amino acid residues of FRs may be substituted so that the reshaped human antibody CDRs form appropriate antigen-binding sites. For example, amino acid sequence mutations may be introduced into FRs using the PCR method used to transplant mouse CDRs into human FRs.

[0265] Desired human antibodies can be obtained by DNA immunization using transgenic animals having all repertoires of human antibody genes (see International Publications WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, and WO1996 / 033735) as immunized animals.

[0266] Furthermore, it is also known to use a human antibody library to obtain a human antibody by panning. For example, the V region of a human antibody is expressed on the surface of a phage in the form of a single-chain antibody (scFv) by a phage display method. A phage expressing an scFv that binds to an antigen can be selected. By analyzing the gene of the selected phage, the DNA sequence encoding the human antibody V region that binds to the antigen can be determined. After determining the DNA sequence of the scFv that binds to the antigen, the V region sequence is fused in frame with the desired human antibody C region sequence, and then an appropriate expression vector is inserted, thereby an expression vector can be made. The expression vector is introduced into the appropriate expression cell listed above, and by expressing the gene encoding the human antibody, the human antibody can be obtained. These methods are already known (see International Publications WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, and WO1995 / 015388).

[0267] The variable region constituting the antibody of the present invention may be a variable region that recognizes any antigen.

[0268] In this specification, "antigen" is not particularly limited and may be any antigen. Examples of antigens include: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressins, adiponectin, ADP-ribosyl cyclase-1, aFGF, AGE, ALCAM, ALK, ALK-1, ALK-7, allergens, α1-antichymotrypsin, α-1-antitrypsin, α-synuclein, α-V / β-1 antagonist, aminin, pullulan, amyloid β, amyloid immunoglobulin Protein heavy chain variable region, amyloid immunoglobulin light chain variable region, androgen, ANG, angiotensinogen, angiopoietin ligand-2, anti-Id, antithrombin III, anthrax, APAF-1, APE, APJ, apolipoprotein A1, apolipoprotein serum amyloid A, apolipoprotein-SAA, APP, APRIL, AR, ARC, ART, Artemin, ASPARTIC, atrial natriuretic factor, atrial natriuretic peptide, atrial natriuretic peptide A, atrial natriuretic peptide B, atrial natriuretic peptide C, av / b3 integrin, Axl, B7-1, B7-2, B7-H, BACE, BACE-1, Bacillus anthracis (Bacillus anthracis) defense antigens, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, BcI, BCMA, BDNF, b-ECGF, β-2-microglobulin, β-lactamase, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, B lymphocyte stimulating factor (BIyS), BMP, BMP-2 (BMP-2a), BMP-3 (osteoblastic protein (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 (Bone-derivedneurotrophicfactor), bovine growth hormone, BPDE, BPDE-DNA, BRK-2, BTC, B-lymphocyte adhesion molecule, C10, C1 inhibitor factor, C1q, C3, C3a, C4, C5, C5a (complement 5a), CA125, CAD-8, cadherin-3, calcitonin, cAMP, carbonic anhydrase-IX, carcinoembryonic antigen (CEA), carcinoma-associated antigen (CAA),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 / EL C、CCL2 / MCP-1、CCL20 / MIP-3-α、CCL21 / SLC、CCL22 / MDC、CCL23 / MPIF-1、CCL24 / eotaxin-2、CCL25 / TECK、CCL26 / eotaxin-3、CCL27 / CTACK、CCL28 / MEC、CCL3 / M1P-1-α、CCL3Ll / LD-78-β、CCL4 / MIP-1-β、CCL5 / RANTES、CCL6 / C10、CCL7 / MCP-3、CCL8 / MCP-2、CCL9 / 10 / MTP-1-γ、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 protein), 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, occludin 18, CLC, Clostridium botulinum toxin, Clostridium difficiledifficile 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 / fragmentation factor, CX3CR1, CXCL, CXCL1 / Gro-α, CXCL10, CXCL11 / I-TAC, CXCL12 / SDF-l-α / β, CXCL13 / BCA-1, CXCL14 / BRAK, CXCL15 / Lungkine.CXCL16, CXCL16, CXCL2 / Gro-βCXCL3 / Gro-γ, CXCL3, CXCL4 / PF4, CXCL5 / ENA-78, CXCL6 / GCP-2, CXCL7 / NAP-2, CXC L8 / 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, complement inhibitory factor (decay-promoting factor), delta-like protein ligand 4, des(1-3)-IGF-I (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 (EGF like domain containing protein7), 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, RSV F protein, F10, F11, F12, F13, F5, F9, Factor Ia, Factor IX, Factor Xa, Factor VII, Factor VIII, Factor VIIIc, Fas 、FcαR、FcεRI、FcγIIb、FcγRI、FcγRIIa、FcγRIIIa、FcγRIIIb、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 cell activation protein (FAP), fibroblast growth factor, fibroblast growth factor-10, fibronectin, FL, FLIP, Flt-3, FLT3 ligand, folate receptor, follicle-stimulating hormone (FSH), fractal chemokine (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-α1, GFR-α2, GFR-α3, GF-β1, gH envelope glycoprotein, GITR, glucagon, glucagon receptor, glucagon-like peptide 1 receptor, Glut 4, glutamate carboxypeptidase II, glycoprotein hormone receptor, glycoprotein IIb / IIIa (GPIIb / 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, Helicobacter pylori (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, hepatocyte growth factor, anthrax defense 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 120V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HMGB-1, HRG, Hrk, HSP47, Hsp90, HSVgD glycoprotein, human myocardial globulin, human cytomegalovirus (HCMV), human growth hormone (hGH), human serum albumin, human tissue plasminogen activator (t-PA), huntingtin protein, HVEM, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFN-α, IFN-β, IFN-γ, IgA, IgA receptor, IgE, IGF, IGF binding protein, IGF-1, IGF-1R, IGF-2, IGFBP, IGFR, IL, IL-1, IL-10, IL-10 receptor, IL-11, IL-11 receptor, IL-12, IL-12 receptor, IL-13, IL-13 receptor, IL-15, IL-15 receptor, IL-16, IL-16 receptor, IL-17 、IL-17 receptor, IL-18 (IGIF), IL-18 receptor, IL-1α, IL-1β, IL-1 receptor, IL-2, IL-2 receptor, IL-20, IL-20 receptor, IL-21, IL-21 receptor, IL-23, IL-23 receptor, IL-2 receptor, IL-3, IL-3 receptor, IL-31, IL-31 receptor, IL-3 receptor, IL-4, IL-4 receptor, IL-5, IL-5 receptor, IL-6, IL-6 receptor, IL-7, IL-7 receptor, IL-8, IL-8 receptor, IL-9, IL-9 receptor, immunoglobulin immune complex, immunoglobulin, INF-α, INF-α receptor, INF-β, INF-β receptor, INF-γ, INF-γ receptor, IFN type-I, IFNtype-I receptor, influenza virus, 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 protein, integrin, integrin α2, integrin α3, integrin α4, integrin α4 / β1, integrin α-V / β-3, integrin α-V / β-6, integrin α4 / β7, integrin α5 / β1, integrin α5 / β3, integrin α5 / β6, integrin ασ(αV), integrin αθ, integrin β1, integrin β2, integrin β3(GPIIb-IIIa), IP-10, I-TAC, JE, kallikrein, kallikrein 11, kallikrein 12, kallikrein 14, Kallikrein 15, Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein L1, Kallikrein L2, Kallikrein L3, Kallikrein L4, Kallikrein binding protein, KC, KDR, keratinocyte growth factor (KGF), keratinocyte growth factor-2 (KGF-2), KGF, killer cell immunoglobulin-like receptor, kit ligand (KL), Kit tyrosine kinase, laminin 5, LAMP, LAPP (pullulan, islet amyloid polypeptide), LAP (TGF-1), latency-associated polypeptide, latent TGF-1, latent TGF-1bp1, LBP, LDGF, LDL, LDL receptor, LECT2, Lefty, leptin, luteinizing hormone (leutinizing hormone hormone)(LH), Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, LFA-3 receptor, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphocyte chemotactic factor, lymphotoxin beta receptor, soluble sphingolipid 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 (67a.a.), MDC (69a.a.), megsin, Mer, MET tyrosine kinase receptor family, metalloproteinase, membrane glycoprotein OX2, mesothelin, MGDF receptor, MGMT, MHC (HLA-DR), microbial protein (microbialprotein), 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 factor), mouse gonadotropin-associated peptide, MPIF, Mpo, MSK, MSP, MUC-16, MUC18, mucin (Mud), Müllerian inhibitory substance (MIS), Mug, MuSK, myelin-associated glycoprotein, myeloid precursor cell inhibitory factor-1 (MPIF-I), NAIP, Nanobody, NAP, NAP-2, NCA 90, NCAD, N-cadherin, NCAM, neprilysin, neural cell adhesion molecule, neuroserpin, nerve growth factor (NGF), neurotrophin-3, neurotrophin-4, neurotrophin-6, neuropilin 1, Neurturin, NGF-β, NGFR, NKG20, N-methionyl human growth hormone, nNOS, NO, Nogo-A, Nogo receptor, hepatitis C virus-derived nonstructural protein type 3 (NS3), NOS, Npn, NRG-3, NT, NT-3, NT-4, NTN, OB, OGG1, oncostatin M, OP-2, OPG, OPN, OSM, OSM receptor, osteoinductive factor (osteoinductive factor factor), 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, placental growth factor, placental alkaline phosphatase (PLAP), placental lactogen, plasminogen activator inhibitor-1, platelet-growth factor, plgR, PLP, polyethylene glycol chains of different sizes (polyglycolchain) (e.g., PEG-20, PEG-30, PEG40), PP14, prekallikrein, prion protein, procalcitonin, programmed cell death protein 1, proinsulin, prolactin, protein 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 factor, 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, T CA-3, T cell receptor α / β, TdT, TECK, TEM1, TEM5, TEM7, TEM8, tenascin, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-β pan-specific (TGF-βPanSpecific), TGF-βRII, TGF-βRIIb, TGF-βRIII, TGF-βRl (ALK-5), TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, TGF-I, thrombin, thrombopoietin (TPO), thymic stromal lymphopoietin (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-α, TNF-β, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2 / DR4), TNFRSF10B (TRAILR2 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 RFN14), 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 TNFRIII / 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 (4-1BB CD137 / ILA), TNFRST23 (DcTRAIL R1 TNFRH1), TNFSF10 (TRAIL Apo-2 ligand / TL2), TNFSF11 (TRANCE / RANK ligand ODF / OPG ligand), TNFSF12 (TWEAK Apo-3 ligand / DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS / TALL1 / THANK / TNFSF20), TNFSF14 (LIGHT HVEM ligand / LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand / TL6), TNFSF1A (TNF-a Conectin / DIF / TNFSF2), TNFSF1B (TNF-bLTa / 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 (4-1BB 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-α and TGF-β, 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 sugar, 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, vitamin B12 receptor, vitronectin receptor, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor (vWF), WIF-1, WNT1, WNT10A, WNT10B, WNT11, WNT16, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, XCL1, XCL2 / SCM-1-β, XCL1 / lymphocyte chemoattractant peptide, XCR1, XEDAR, XIAP, XPD, etc.

[0269] In the two variable regions of the antibody contained in the antigen-binding molecule of the present invention, the "first antigen" and "second antigen" bound by the variable regions that can bind to two different antigens but cannot bind to these antigens simultaneously are preferably, for example, antigens expressed on immune cell surface molecules (e.g., T cell surface molecules, NK cell surface molecules, dendritic cell surface molecules, B cell surface molecules, NKT cell surface molecules, MDSC cell surface molecules, macrophage surface molecules), tumor cells, tumor blood vessels, stromal cells, etc., but also expressed in normal tissues (integrin, tissue factor, VEGFR, PDGFR, EGFR, IGFR, MET chemokine receptor, heparan sulfate proteoglycan, CD44, fibronectin, DR5, TNFRSF, etc.). As a combination of the "first antigen" and the "second antigen", one of the first antigen and the second antigen is preferably a molecule specifically expressed on T cells, and the other antigen is preferably a molecule expressed on the surface of T cells or other immune cells. As another scheme, as a combination of "the first antigen" and "the second antigen", either of the first antigen and the second antigen is preferably a molecule specifically expressed on T cells, for example, and the other antigen is preferably expressed on immune cells, and is a different molecule from the previously selected antigen. Specifically, for example, as molecules specifically expressed on T cells, CD3 and T cell receptors can be cited. CD3 is particularly preferred. As the site of CD3 to which the antigen-binding molecule of the present invention binds, for example, in the case of human CD3, as long as it is an epitope present in the γ chain, δ chain or ε chain sequence constituting human CD3, it can bind to any epitope. An epitope present in the extracellular region of the ε chain of the human CD3 complex is particularly preferred. The structure of the γ chain, δ chain or ε chain constituting CD3, the polynucleotide sequence thereof is described in SEQ ID NO: 83 (NM_000073.2), 85 (NM_000732.4) and 87 (NM_000733.3), and the polypeptide sequence thereof is described in SEQ ID NO: 84 (NP_000064.1), 86 (NP_000723.1) and 88 (NP_000724.1) (RefSeq accession numbers are shown in parentheses). Furthermore, examples of the other antigen include: Fcγ receptors, TLRs, lectins, IgA, immune restriction site molecules, TNF superfamily molecules, TNFR superfamily molecules, and NK receptor molecules.

[0270] In addition, of the two variable regions of the antibody contained in the antigen-binding molecule of the present invention, the "third antigen" different from the previous "first antigen" and "second antigen" to which the other variable region binds is preferably a tumor cell-specific antigen, which includes, in addition to antigens expressed with the malignant transformation of cells, abnormal sugar chains presented on the cell surface or protein molecules when cells become cancerous. Specifically, 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-associated antigen p97, melanoma antigen gp75, high molecular weight melanoma antigen (HMW-MAA), prostate-specific membrane antigen, carcinoembryonic antigen (CEA), polymorphic epithelial mucin antigen, human milk fat globule antigen, CEA, TAG-72, CO17-1A, GICA Colorectal tumor-associated antigens such as 19-9, CTA-1 and LEA, Burkitt's 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 tumor viruses and RNA tumor viruses, CEA of the colon, 5T4 carcinoma fetal trophoblast glycoprotein and bladder tumor carcinoma fetal antigen α-fetoprotein, differentiation antigens such as human lung cancer antigen L6 and L20, fibrosarcoma antigen, human leukemia T cell antigen-Gp37, neoglycoprotein, sphingolipids, breast cancer antigens such as EGFR (epithelial growth factor receptor), NY-BR-16, NY-B R-16 and HER2 antigens (p185HER2), polymorphic epithelial mucin (PEM), malignant human lymphocyte antigen-APO-1, differentiation antigens such as I antigens visible in fetal erythrocytes, primitive endoderm I antigens visible in adult erythrocytes, embryos before transplantation, I (Ma) visible in gastric cancer, M18, M39 visible in breast epithelium, SSEA-1, VEP8, VEP9, Myl, VIM-D5 visible in bone marrow cells, D156-22, TRA-1-85 (blood group H) visible in colorectal cancer, SCP-1 visible in testicular and ovarian cancer, C14 visible in colon cancer, F3 visible in lung cancer, AH6 visible in gastric cancer, Y hapten, Ley visible in embryonic carcinoma cells, TL5 (blood group A), EGF receptor visible in A431 cells, E1 series visible in pancreatic cancer (blood group B), FC10 visible in embryonic carcinoma cells.2. Gastric cancer antigens, CO-514 (blood group Lea) seen in adenocarcinoma, NS-10, CO-43 (blood group Leb) seen in adenocarcinoma, G49 seen in EGF receptor of A431 cells, MH2 (blood group ALeb / Ley) seen in colon cancer, 19.9 seen in colon cancer, gastric cancer mucin, T5A7 seen in bone marrow cells, R24 seen in melanoma, 4.2, GD3, D1.1, OFA-1, GM2, OFA-2, GD2, and M1:22:25:8 seen in embryonic carcinoma cells, and SSEA-3 and SSEA-4 seen 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 mutant A, adenocarcinoma antigen ART1, tumor Tumor-associated brain-testicular cancer antigens (cancer neuron antigen MA2, tumor-associated neuron antigen), neuronal oncogenous abdominal antigen 2 (NOVA2), blood cell cancer antigen gene 520, tumor-associated antigen CO-029, tumor-associated antigen MAGE-C1 (cancer / testis antigen CT7), MAGE-B1 (MAGE-XP antigen), MAGE-B2 (DAM6), MAGE-2, MAGE-4a, MAGE-4b and MAGE-X2, cancer testis antigen (NY-EOS-1), YKL-40 and any fragments of the above polypeptides or modified structures thereof (the above modified phosphate groups or sugar chains, etc.), EpCAM, EREG, CA19-9, CA15-3, sialylated SSEA-1 (SLX), HER2, PSMA, CEA, CLEC12A, etc.

[0271] The antigen binding molecules of the present invention can be prepared by methods well known to those skilled in the art. For example, antibodies can be made by the following methods, but are not limited thereto. It is known that a plurality of combinations of host cells and expression vectors are used to introduce genes encoding isolated polypeptides into appropriate hosts to make antibodies. These expression systems can all be used to separate 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 appropriately. Specifically, animal cells can exemplify the following cells.

[0272] (1) Mammalian cells: CHO (Chinese hamster ovary cell line), COS (Monkey kidney cell line), myeloma cells (Sp2 / O, NS0, etc.), BHK (baby hamster kidney cell line), HEK293 (human embryonic kidney cell line with sheared adenovirus (Ad) 5 DNA), PER.C6 cells (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))

[0273] (2) Amphibian cells: Xenopus laevis oocytes, etc.

[0274] (3) Insect cells: sf9, sf21, Tn5, etc.

[0275] In addition, antibodies can also be produced using E. coli (mAbs 2012 Mar-Apr; 4(2):217-225.) or yeast (WO2000023579). Antibodies produced using E. coli do not have sugar chains added. Antibodies produced using yeast do have sugar chains added.

[0276] The DNA encoding the heavy chain of the antibody and the heavy chain DNA encoding one or more amino acid residues in the variable region are substituted with other amino acids of the target, and the DNA encoding the light chain of the antibody are expressed. The DNA encoding the heavy chain or light chain in which one or more amino acid residues in the variable region are substituted with other amino acids of the target can be obtained, for example, as follows: DNA encoding the variable region of the antibody prepared using a known method for a certain antigen is obtained, and the codon encoding a specific amino acid in the region is introduced into the region to encode the other amino acid of the target.

[0277] In addition, a DNA encoding a protein in which one or more amino acid residues in the variable region of an antibody prepared using a known method for a certain antigen are substituted with other amino acids of the target is designed in advance, and by chemically synthesizing the DNA, a DNA encoding a heavy chain in which one or more amino acid residues in the variable region are substituted with other amino acids of the target can also be obtained. There are no particular limitations on the amino acid substitution site and substitution type. As preferred regions for amino acid changes, solvent-exposed regions and loop regions in the variable region can be cited. Among them, CDR1, CDR2, CDR3, FR3 regions, and loop regions are preferred. Specifically, Kabat numbers 31 to 35, 50 to 65, 71 to 74, 95 to 102 of the H chain variable region are preferred, and Kabat numbers 24 to 34, 50 to 56, 89 to 97 of the L chain variable region are preferred, and Kabat numbers 31, 52a to 61, 71 to 74, 97 to 101 of the H chain variable region are more preferred, and Kabat numbers 24 to 34, 51 to 56, 89 to 96 of the L chain variable region are more preferred.

[0278] Furthermore, amino acid changes are not limited to substitutions, and may be deletions, additions, insertions, or modifications, or a combination thereof.

[0279] In addition, 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 divided into partial DNAs for preparation. As a combination of partial DNAs, for example, DNA encoding a variable region and a DNA encoding a constant region, or a DNA encoding a Fab region and a DNA encoding an Fc region, etc. can be cited, but are not limited to these combinations. In addition, DNA encoding a light chain can also be divided into partial DNAs for preparation.

[0280] As a method for expressing the above-mentioned DNA, the following method can be cited. For example, the DNA encoding the heavy chain variable region and the DNA encoding the heavy chain constant region are integrated (recombined) into an expression vector to construct a heavy chain expression vector. Similarly, the DNA encoding the light chain variable region and the DNA encoding the light chain constant region are integrated into an expression vector to construct a light chain expression vector. The genes of these heavy chains and light chains can also be integrated into a single vector.

[0281] When the DNA encoding the target antibody is integrated into an expression vector, it is expressed under the control of an expression control region, such as an enhancer or a promoter and integrated into the expression vector. Next, the host cell is transformed with the expression vector to express the antibody. At this time, a combination of an appropriate host and an expression vector can be used.

[0282] Examples of vectors include M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script, etc. Furthermore, for the purpose of subcloning or excision of cDNA, in addition to the above-mentioned vectors, for example, pGEM-T, pDIRECT, pT7, etc. can also be used.

[0283] When a vector is used for the purpose of producing the antibody of the present invention, an expression vector is particularly useful. For example, when the host is Escherichia coli such as JM109, DH5α, HB101, XL1-Blue, etc., it is indispensable to have a promoter that can effectively express in Escherichia coli, such as lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427, the entire contents of which are incorporated herein by reference), araB promoter (Better et al., Science (1988) 240, 1041-1043, the entire contents of which are incorporated herein by reference), or T7 promoter. Examples of such vectors include pGEX-5X-1 (manufactured by Pharmacia), "QIAexpress system" (manufactured by QIAGEN), pEGFP, or pET (in this case, the host is preferably BL21 expressing T7 RNA polymerase).

[0284] In addition, the vector may also contain a signal sequence for polypeptide secretion. As a signal sequence for polypeptide secretion, when produced in the periplasm of Escherichia coli, the pelB signal sequence (Lei, SP et al. J. Bacteriol. (1987) 169, 4397, the entire contents of which are incorporated herein by reference) can be used. The introduction of the vector into the host cell can be carried out using, for example, the liposome method, the calcium phosphate method, or the DEAE-dextran method.

[0285] In addition to the Escherichia coli expression vector, examples of the vector for producing the polypeptide of the present invention include: expression vectors derived from mammals (e.g., pcDNA3 (manufactured by Invitrogen) or pEGF-BOS (Nucleic Acids. Res. 1990, 18 (17), p5322, the entire contents of which are incorporated herein by reference), pEF, pCDM8), expression vectors derived from insect cells (e.g., "Bac-to-BAC baculovirus expression system" (manufactured by GIBCOBRL), pBacPAK8), expression vectors derived from plants (e.g., pMH1, pMH2), expression vectors derived from animal viruses (e.g., pHSV, pMV, pAdexLcw), expression vectors derived from retroviruses (e.g., pZIPneo), expression vectors derived from yeast (e.g., "Pichia Expression Kit" (manufactured by Invitrogen), pNV11, SP-Q01), and expression vectors derived from Bacillus subtilis (e.g., pPL608, pKTH50).

[0286] When the purpose is to express in animal cells such as CHO cells, COS cells, NIH3T3 cells, HEK293 cells, etc., it is indispensable to have a promoter necessary for expression in the cells, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108, the entire contents of which are incorporated herein by reference), MMTV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322, the entire contents of which are incorporated herein by reference), CAG promoter (Gene. (1991) 108, 193, the entire contents of which are incorporated herein by reference), CMV promoter, etc., and it is further preferred to have a gene for selecting transformed cells (for example, a drug resistance gene 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, EBNA1 protein may be co-expressed for the purpose of increasing the copy number of a gene, and in such cases, a vector having the replication origin OriP is used (Biotechnol Bioeng. 2001 Oct 20; 75(2): 197-203., Biotechnol Bioeng. 2005 Sep 20; 91(6): 670-7.).

[0287] Furthermore, in the case of stably expressing the gene and amplifying the gene copy number in the cell, the method of introducing a vector (e.g., pCHOI, etc.) having a complementary DHFR gene into CHO cells lacking the nucleic acid synthesis pathway and amplifying by methotrexate (MTX) can be cited. In addition, in the case of transient expression of the gene, the method of transforming COS cells holding a gene expressing SV40 T antigen on the chromosome with a vector (pcD, etc.) holding the replication origin of SV40 can be cited. As the replication origin, the replication origin derived from polyoma virus, adenovirus, bovine papilloma virus (BPV), etc. can also be used. Furthermore, in order to amplify the gene copy number in the host cell line, the expression vector can contain aminoglycoside phosphotransferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthine guanine phosphoribosyl transferase (Ecogpt) gene, dihydrofolate reductase (dhfr) gene, etc. as a selection marker.

[0288] The recovery of the antibody can be carried out, for example, by separating it from the cells or culture medium of the molecularly transformed cells after culturing the transformed cells. For the separation and purification of the antibody, centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, C1q, FcRn, protein A column, protein G column, affinity column chromatography, ion exchange column chromatography, gel filtration chromatography and the like can be appropriately combined.

[0289] As an efficient method for producing multispecific antibodies, the above-mentioned techniques such as Knobs-into-holes technology (WO1996 / 027011, Ridgway JB et al., Protein Engineering (1996) 9, 617-621, Merchant AM et al. Nature Biotechnology (1998) 16, 677-681) or the technique of introducing charge repulsion to inhibit the association between non-target H chains (WO2006 / 106905) can be used.

[0290] Furthermore, the present invention provides a method for preparing an antigen-binding molecule of the present invention, wherein the antigen-binding molecule comprises: a variable region (first variable region) that can bind to two different first antigens and second antigens and does not bind to the first antigen and the second antigen at the same time, and a variable region (second variable region) that binds to a third antigen different from the first antigen and the second antigen, the method comprising: a step of preparing an antigen-binding molecule library with diversified amino acid sequences of the first variable region.

[0291] For example, a preparation method comprising the following steps can be mentioned:

[0292] Step (i): preparing a library of antigen-binding molecules, wherein at least one amino acid in the variable region of an antibody binding to a first antigen or a second antigen is changed, and the library comprises variable regions in which at least one amino acid in the changed variable region is different from each other;

[0293] Step (ii): selecting, from the prepared library, an antigen-binding molecule comprising a variable region that has binding activity to the first antigen and the second antigen but does not bind to the first antigen and the second antigen simultaneously;

[0294] Step (iii): culturing a host cell 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, so that the antigen-binding molecule containing the variable region of an antibody that binds to the first antigen and the second antigen but not to the first antigen and the second antigen simultaneously, and / or the variable region that binds to the third antigen is expressed; and

[0295] Step (iv): recovering the antigen-binding molecules from the above host cell culture.

[0296] It should be noted that, in the present preparation method, step (ii) may be the following optional step:

[0297] Step (v): Selecting, from the prepared library, antigen-binding molecules comprising variable regions that have binding activity to the first antigen and the second antigen but do not simultaneously bind to the first antigen and the second antigen expressed on different cells.

[0298] The antigen-binding molecule used in step (i) above is not particularly limited as long as it contains an antibody variable region, and may be an antibody fragment such as Fv, Fab, or Fab', or an antibody containing an Fc region.

[0299] As the amino acid to be modified, for example, in the variable region of the antibody that binds to the first antigen or the second antigen, an amino acid that does not lose the binding to the antigen by amino acid modification is selected.

[0300] The amino acid modifications of the present invention may be used alone or in combination of two or more.

[0301] When multiple combinations are used, the number of combinations is not particularly limited, and is, for example, 2 to 30, preferably 2 to 25, 2 to 22, 2 to 20, 2 to 15, 2 to 10, 2 to 5, 2 to 3.

[0302] In the case of multiple combinations, the amino acid change can be added only to the heavy chain variable region or the light chain variable region of the antibody, or the amino acid change can be appropriately distributed and added to both the heavy chain variable region and the light chain variable region as the preferred region for amino acid change, which can be cited as: solvent exposed region and loop region in the variable region. Among them, CDR1, CDR2, CDR3, FR3 region, loop region are preferred. Specifically, Kabat numbers 31 to 35, 50 to 65, 71 to 74, 95 to 102 of the H chain variable region are preferred, and Kabat numbers 24 to 34, 50 to 56, 89 to 97 of the L chain variable region are preferred, and Kabat numbers 31, 52a to 61, 71 to 74, 97 to 101 of the H chain variable region are more preferred, and Kabat numbers 24 to 34, 51 to 56, 89 to 96 of the L chain variable region are more preferred.

[0303] In addition, adding changes in amino acid residues also includes the following situations: in the variable region of an antibody combined with the first antigen or the second antigen, randomly changing the amino acid in the above-mentioned region, or inserting a peptide known in advance to have binding activity to a desired antigen in the above-mentioned region. In this way, from the antigen binding molecules to which the changes have been added, select a variable region that can be combined with the first antigen and the second antigen, but cannot be combined with these antigens at the same time, thereby the antigen binding molecules of the present invention can be obtained. As an example of a peptide known in advance to have binding activity to a desired antigen, the peptide shown in the above-mentioned Table 1 can be enumerated.

[0304] Whether it is a variable region that can bind to the first antigen and the second antigen but cannot bind to these antigens at the same time, and whether it is a variable region that can bind to both the first antigen and the second antigen at the same time when either one of the first antigen and the second antigen is present on the 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 at the same time when each is expressed on different cells can be confirmed in the same manner as the above method.

[0305] Furthermore, the present invention also provides a method for preparing the antigen-binding molecule of the present invention, wherein the antigen-binding molecule comprises: an antibody variable region (first variable region) that can bind to two different first antigens and second antigens and does not bind to the first antigen and the second antigen at the same time, and the preparation method comprises: a step of preparing an antigen-binding molecule library with diversified amino acid sequences of the first variable region.

[0306] As a method for preparing such an antigen-binding molecule, for example, there can be mentioned a method comprising the following steps: step (i): preparing a library of antigen-binding molecules, wherein at least one amino acid in the variable region of an antibody binding to a first antigen or a second antigen is changed, and the library comprises variable regions in which at least one amino acid in the changed variable region is different from each other;

[0307] Step (ii): selecting, from the prepared library, an antigen-binding molecule comprising a variable region that has binding activity to the first antigen and the second antigen but does not bind to the first antigen and the second antigen simultaneously;

[0308] Step (iii): culturing a nucleic acid encoding the variable region of the antigen-binding molecule selected in step (ii) to express an antigen-binding molecule containing an antibody variable region that can bind to the first antigen and the second antigen but not to the first antigen and the second antigen simultaneously; and

[0309] Step (iv): recovering the antigen-binding molecules from the above host cell culture.

[0310] It should be noted that as preferred regions for the above-mentioned amino acid changes, the heavy chain variable region can be cited. Further preferred regions include: solvent-exposed regions and loop regions in the variable region. Among them, CDR1, CDR2, CDR3, FR3 regions, and loop regions are preferred. Specifically, Kabat numbers 31 to 35, 50 to 65, 71 to 74, 95 to 102 of the H chain variable region are preferred, and Kabat numbers 24 to 34, 50 to 56, 89 to 97 of the L chain variable region are preferred, and Kabat numbers 31, 52a to 61, 71 to 74, 97 to 101 of the H chain variable region are more preferred, and Kabat numbers 24 to 34, 51 to 56, 89 to 96 of the L chain variable region are more preferred.

[0311] It should be noted that, in the present preparation method, the above step (ii) may be the following optional step:

[0312] Step (v): Selecting, from the prepared library, antigen-binding molecules containing variable regions that have binding activity to the first antigen and the second antigen but do not simultaneously bind to the first antigen and the second antigen expressed on different cells.

[0313] The antigen-binding molecule used in step (i) above is not particularly limited as long as it contains an antibody variable region, and may be an antibody fragment such as Fv, Fab, or Fab', or an antibody containing an Fc region.

[0314] As the amino acid to be modified, for example, in the variable region of the antibody that binds to the first antigen or the second antigen, an amino acid that does not lose the binding to the antigen by amino acid modification is selected.

[0315] The amino acid modifications of the present invention may be used alone or in combination of two or more.

[0316] When multiple combinations are used, the number of combinations is not particularly limited, and is, for example, 2 to 30, preferably 2 to 25, 2 to 22, 2 to 20, 2 to 15, 2 to 10, 2 to 5, 2 to 3.

[0317] In the case of multiple combinations, the amino acid changes may be added to only one of the heavy chain variable region or the light chain variable region of the antibody, or the amino acid changes may be added to both the heavy chain variable region and the light chain variable region in an appropriately distributed manner.

[0318] In addition, the addition of changes to the amino acid residues also includes the following situations: in the variable region of the antibody that binds to the first antigen or the second antigen, the amino acids in the above region are randomly changed; or, a peptide that is known in advance to have binding activity to the desired antigen is inserted into the above region. In this way, a variable region that can bind to the first antigen and the second antigen but cannot bind to these antigens at the same time is selected from the antigen-binding molecules to which the changes are added, thereby obtaining the antigen-binding molecules of the present invention. As examples of peptides that are known in advance to have binding activity to the desired antigen, the peptides shown in Table 1 above can be cited.

[0319] Whether it is a variable region that can bind to the first antigen and the second antigen but cannot bind to these antigens at the same time, and whether it is a variable region that can bind to both the first antigen and the second antigen at the same time when either one of the first antigen and the second antigen is present on the 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 at the same time when each is expressed on different cells can be confirmed in the same manner as the above method.

[0320] Furthermore, antigen-binding molecules produced by this production method are also included in the present invention.

[0321] There is no particular limitation on the type or scope of the amino acid changes introduced by the present method.

[0322] One non-limiting embodiment of the library of the present invention is a library in which CD3 (when human CD3, the γ chain, δ chain, or ε chain constituting human CD3) is selected as the first antigen and comprises antigen-binding molecules that bind to CD3 and an arbitrary second antigen.

[0323] In this specification, "library" refers to a plurality of antigen binding molecules or a plurality of fusion polypeptides containing antigen binding molecules, or nucleic acids or polynucleotides encoding these sequences. The sequences of a plurality of antigen binding molecules or a plurality of fusion polypeptides containing antigen binding molecules contained in the library are not single sequences, but antigen binding molecules or fusion polypeptides containing antigen binding molecules having different sequences.

[0324] In one embodiment of the present invention, a fusion polypeptide of an antigen-binding molecule of the present invention and a heterologous polypeptide can be prepared. In a certain embodiment, the fusion polypeptide can be fused with at least a portion of a viral coat protein, for example, selected from pIII, pVIII, pVII, pIX, Soc, Hoc, gpD, pVI and mutants thereof.

[0325] In a certain embodiment, the antigen binding molecules of the present invention can be ScFv, Fab fragments, F(ab)2 or F(ab')2, so in another embodiment, a library is provided, the library is mainly composed of a variety of fusion polypeptides that are fusion polypeptides of these antigen binding molecules and heterologous polypeptides and sequences are different from each other. Specifically, a library is provided that is mainly composed of a variety of fusion polypeptides obtained by fusion of at least a portion of these antigen binding molecules and viral coat proteins, for example, selected from pIII, pVIII, pVII, pIX, Soc, Hoc, gpD, pVI and mutants thereof, and sequences are different from each other. The antigen binding molecules of the present invention can also include a dimerization domain. In a certain embodiment, the above-mentioned dimerization domain may be present between the variable region of the heavy chain or light chain of the antibody and at least a portion of the viral coat protein. In the dimerization domain, at least one dimerization sequence and / or a sequence containing 1 or more cysteine ​​residues may be included. The dimerization domain is preferably capable of being connected to the C-terminus of the heavy chain variable region or the constant region. The dimerization domain may have various structures depending on whether the antibody variable region is prepared as a fusion polypeptide component with a viral coat protein component (without an amber stop codon after the dimerization domain), or whether the antibody variable region is prepared without mainly containing a viral coat protein component (for example, with an amber stop codon after the dimerization domain). When the antibody variable region is prepared mainly as a fusion polypeptide with a viral coat protein component, bivalent expression is achieved by one or more disulfide bonds and / or a single dimerization sequence.

[0326] In this specification, the term "sequences are different from each other" in the description of multiple antigen-binding molecules with different sequences means that the sequences of the antigen-binding molecules in the library are different from each other. That is, the number of different sequences in the library reflects the number of independent clones with different sequences in the library, and is sometimes also regarded as the "library size". The size of a common phage display library is 10 6 Up to 10 12 By using well-known techniques such as ribosome display, the library size can be enlarged to 10 14 However, the actual number of phage particles used in panning selection of phage libraries is usually 10 to 10,000 times larger than the library size. This excess multiple is also called "library equivalent number", which means that there can be 10 to 10,000 clones with the same amino acid sequence. Therefore, the term "sequences are different from each other" in the present invention means that the sequences of the antigen-binding molecules in the library excluding the library equivalent number are the same, and more specifically means that there are 10 6 Up to 10 14 10, preferably 10 7 Up to 10 12 More preferably, 10 8 Up to 10 11 10, particularly preferably 8 Up to 10 10 The sequences of the antigen-binding molecules are different from each other.

[0327] Furthermore, the expression "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 with different binding activities to the first and / or second antigens among the number of independent clones with different sequences in the library. Specifically, it is preferred that the antigen-binding molecules showing such binding activity exist in at least 10 of the libraries. 4 Furthermore, it is more preferred that the antigen-binding molecules provided by the present invention exhibit such binding activity in the presence of at least 10 5 More preferably, the present invention provides a library of antigen-binding molecules showing such binding activity. 6 It is particularly preferred that the present invention provide a library of at least 10 antigen-binding molecules showing such binding activity. 7 It is also preferred that the present invention provide a library of at least 10 antigen-binding molecules showing such binding activity. 8In other expressions, it can also be appropriately expressed as the ratio of antigen-binding molecules with different binding activities for the first and / or second antigens among the number of independent clones with different sequences in the library. Specifically, the present invention provides a library in which the antigen-binding molecules showing such binding activity account for 0.1% to 80%, preferably 0.5% to 60%, more preferably 1% to 40%, further preferably 2% to 20%, and particularly preferably 4% to 10% of the number of independent clones with different sequences in the library. The situation of fusion polypeptides, polynucleotide molecules or vectors is also the same as above, and can be expressed by the number of molecules or the ratio of all molecules. In addition, the situation of viruses is also the same as above, and can be expressed by the number of virus individuals or the ratio of all individuals.

[0328] In this specification, "phage display" refers to a method of presenting mutant polypeptides in the form of proteins obtained by fusion with at least a portion of the coat protein on the surface of a phage, such as a filamentous phage particle. The effectiveness of phage display lies in that sequences that bind to the target antigen with high affinity can be quickly and efficiently selected from a large library of randomized protein mutants. The presentation of peptide and protein libraries on phage is used to screen millions of polypeptides involving specific binding properties. Multivalent phage display methods are used to present small random peptides and small proteins by fusion with gene III or gene VIII of filamentous phage (Wells and Lowman (Curr. Opin. Struct. Biol. (1992) 3, 355-362) and references therein). In monovalent phage display, a library of proteins or peptides is fused with gene III or a portion thereof, expressed at low levels in the presence of native gene III protein, so that the phage particles present 1 or 0 copies of the fusion protein. Since the affinity effect is lower than that of polyvalent phage, phagemid vectors are used in selection based on the affinity of endogenous ligands, but this vector simplifies DNA manipulation (Lowman and Wells, Methods: A Companion to Methods in Enzymology (1991) 3, 205-216).

[0329] "Phagemid" refers to a plasmid vector having a bacterial replication origin, such as ColE1, and a copy of the intergenic region of the phage. Phagemids can also appropriately use any known phage, such as phagemids of filamentous phages and lambda phages. Plasmids usually also contain a selection marker for antibiotic resistance. DNA fragments cloned into these vectors can be propagated as plasmids. When cells introduced with these vectors have all the genes necessary for producing phage particles, the replication mode of the plasmid changes to rolling circle replication, generating copies of one chain of the plasmid DNA and packaging phage particles. Phagemids can form infectious or non-infectious phage particles. This term includes a phagemid that contains a phage coat protein gene or a fragment thereof that is combined with the gene of a heterologous polypeptide by genetic fusion in a manner that the heterologous polypeptide is presented on the surface of the phage particle.

[0330] The term "phage vector" refers to a double-stranded replicative type of a bacteriophage that contains a heterologous gene and is capable of replication. The phage vector has a phage replication origin that can replicate the phage and form phage particles. The phage is preferably a filamentous phage, such as M13, f1, fd, Pf3 phage or a derivative thereof, or a lambda phage, such as lambda, 21, phi80, phi81, 82, 424, 434, others, or a derivative thereof.

[0331] "Oligonucleotide" refers to a relatively short single-stranded or double-stranded polydeoxynucleotide chemically synthesized by a known method (e.g., solid phase method, such as phosphotriester, phosphite, or phosphoramidite chemistry using the method described in EP266032, or by the method of deoxynucleotide H-phosphonate intermediates described in Froeshler et al. (Nucl. Acids. Res. (1986) 14, 5399-5407). Other methods include the polymerase chain reaction described below and other automatic primer methods, and oligonucleotide synthesis on a solid support. All of these methods are described in Engels et al. (Agnew. Chem. Int. Ed. Engl. (1989) 28, 716-734). These methods are used if the entire nucleic acid sequence of the 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, the individual amino acid residues are known, and using the preferred coding residues, the possible nucleic acid sequence can be appropriately inferred. The oligonucleotide can be purified by polyacrylamide gel or molecular weight column, or by precipitation.

[0332] The terms "fusion protein" and "fusion polypeptide" refer to a polypeptide having two parts bound to each other by a covalent bond, each part having different properties. The property may be, for example, a biological property such as in vitro or in vivo activity. In addition, the property may even be a single chemical or physical property, such as binding to a target antigen, catalysis of a reaction, etc. The two parts may be bound directly by a single peptide bond, or via a peptide linker comprising one or more amino acid residues. Typically, the two parts and the linker are present in the same reading frame. Preferably, the two parts of the polypeptide are obtained from heterologous or different polypeptides.

[0333] The term "coat protein" refers to a protein at least a portion of which is present on the surface of a virus particle. From a functional perspective, a coat protein is any protein that binds to a virus particle during the construction of the virus in a host cell, and the virus binds to it until it infects other host cells. A coat protein can be either a major coat protein or a minor coat protein. A minor coat protein generally refers to a coat protein present in the viral coat, preferably at least about 5, more preferably at least about 7, more preferably at least about 10 or more copies of the protein are present in each virion. A major coat protein can exist in tens, hundreds or thousands of copies in each virion. As an example of a major coat protein, the p8 protein of a filamentous bacteriophage can be cited.

[0334] As a non-limiting embodiment of the present invention, the following six methods for preparing libraries are exemplified.

[0335] Method 1: Inserting a peptide (this term is used to include both polypeptides and proteins) that binds to a second antigen into an antigen-binding molecule that binds to a first antigen;

[0336] Method 2: A library is prepared so that various amino acids are presented at sites that can lengthen (extend) the loops in the antigen-binding molecule, and antigen-binding molecules having binding activity to an arbitrary second antigen are obtained from the library using antigen-binding activity as an indicator;

[0337] Method 3: Using antibodies produced by site-directed mutagenesis, amino acids that maintain binding activity to the first antigen are identified from antigen-binding molecules known to bind to the first antigen, and then, using the antigen-binding activity as an indicator, antigen-binding molecules that have binding activity to an arbitrary second antigen are obtained from the library of the identified amino acids;

[0338] Method 4: In method 3, an antibody library is further prepared so that various amino acids are presented at sites that can lengthen (extend) the loop in the antigen-binding molecule, and antigen-binding molecules having binding activity to an arbitrary second antigen are obtained from the library using antigen-binding activity as an indicator;

[0339] Method 5: In method 1, 2, 3 or 4, a change is made so that a sequence to which a sugar chain is added (e.g., NxS, NxT, wherein x is an amino acid other than P) is presented, and a sugar chain recognized by a sugar chain receptor is added (e.g., a high mannose type sugar chain is added, which is recognized by a high mannose receptor. It is known that a high mannose type sugar chain is obtained by adding kifnosine during antibody expression (MAbs. 2012 Jul-Aug; 4(4): 475-87));

[0340] Method 6: In method 1, 2, 3 or 4, Cys, Lys or a non-natural amino acid is inserted or substituted at a loop site or a site that can be changed to various amino acids, and a domain that binds to a second antigen is added by covalent bonding (this is a method represented by an antibody drug conjugate, which is a method of binding to Cys, Lys or a non-natural amino acid by covalent bonding (mAbs 6: 1, 34-45; January / February 2014; WO2009 / 134891A2; Bioconjug Chem. 2014Feb 19; 25(2): 351-61))

[0341] It should be noted that in the six library preparation methods exemplified above, the site of replacing the amino acid in the antigen binding molecule or the site of inserting the peptide into the antigen binding molecule is preferably the Fab or variable region portion of the antigen binding molecule. As preferred regions, solvent exposed regions and loop regions in the variable region can be cited. Among them, CDR1, CDR2, CDR3, FR3 regions, and loop regions are preferred. Specifically, Kabat numbers 31 to 35, 50 to 65, 71 to 74, 95 to 102 of the H chain variable region are preferred, and Kabat numbers 24 to 34, 50 to 56, 89 to 97 of the L chain variable region are preferred, and Kabat numbers 31, 52a to 61, 71 to 74, 97 to 101 of the H chain variable region are more preferred, and Kabat numbers 24 to 34, 51 to 56, 89 to 96 of the L chain variable region are more preferred.

[0342] As one embodiment of the method for inserting a peptide that binds to a second antigen into an antigen-binding molecule that binds to a first antigen as described in the above method 1., another embodiment includes the method of inserting G-CSF as exemplified in Angew Chem Int Ed Engl. 2013 Aug 5; 52(32):8295-8. As another embodiment, the peptide to be inserted can be obtained from a library presenting peptides, or a naturally occurring protein in its entirety or in part can be used.

[0343] In order to identify amino acids that maintain binding activity to CD3 (in the case of human CD3, the γ chain, δ chain, or ε chain constituting human CD3) as the first antigen, for example, amino acid changes can be made at sites believed to be involved in antigen binding, and an antibody with amino acid changes can be prepared for determination. In the evaluation of CD3 binding of an antibody with amino acid changes, a method known to those skilled in the art can be appropriately selected, for example, ELISA or FACS (fluorescence activated cell sorting), ALPHA screening (Amplified Luminescent Proximity Homogeneous Assay), or BIACORE method using surface plasmon resonance (SPR) phenomenon can be used for measurement.

[0344] When identifying amino acids that maintain binding activity to CD3, for example, the results of the ratio of the binding amounts of various variants can be used for the antibody before the change. That is, the value of Z (ratio of binding amount) = Y / X can be used when the binding amount of the antibody before the change is X and the binding amount of one amino acid variant is Y. When Z (ratio of binding amount) is 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, and preferably 0.8 or more, it can be considered that the binding to the antibody before the change is maintained. Antibody libraries can be prepared to present amino acids that maintain these bindings.

[0345] ECM (Extracellular matrix) is one of the components outside cells and exists in various parts of the body. Therefore, it is known that antibodies that strongly bind to ECM will have poor blood dynamics (shortened half-life) (WO2012093704A1). Therefore, amino acids that do not enhance ECM binding are also preferred for the amino acids presented in the antibody library.

[0346] When selecting amino acids that do not enhance ECM binding, for example, the ECM binding is evaluated according to the method of Reference Example 2, and the value obtained by dividing the ECM binding value (ECL response) of each variant 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 ECM binding enhancement effect produced by multiple changes, the value can be 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, and up to 30 times as an effective value, but preferably up to 10 times is used as an effective value for the library. Antibody libraries can be prepared to represent the amino acids selected in this way.

[0347] It should be noted that, although not limited to this, when the peptide insertion in CDR3 is 6 amino acids, if the extended loop of CDR3 contains multiple amino acids with positively charged side chains, the binding to the ECM is enhanced, so it is preferred that amino acids with more than 3 positively charged side chains are not present in the loop.

[0348] The library of the present invention can insert peptides into the variable region in order to increase the diversity of the library. Preferred regions for peptide insertion include: solvent-exposed regions and loop regions in the variable region. Among them, CDR1, CDR2, CDR3, FR3 regions, and loop regions are preferred. Specifically, Kabat numbers 31 to 35, 50 to 65, 71 to 74, 95 to 102 of the H chain variable region, and Kabat numbers 24 to 34, 50 to 56, and 89 to 97 of the L chain variable region are preferred, and Kabat numbers 31, 52a to 61, 71 to 74, and 97 to 101 of the H chain variable region are more preferred, and Kabat numbers 24 to 34, 51 to 56, and 89 to 96 of the L chain variable region are more preferred. The region with Kabat numbers 99-100 of the H chain variable region is further preferred. In addition, when the amino acids are changed, amino acid combinations that improve the binding activity to the antigen can be introduced.

[0349] As a non-limiting embodiment of the present invention, the length of the peptide to be inserted can be: 1 to 3 amino acids, 4 to 6 amino acids, 7 to 9 amino acids, 10 to 12 amino acids, 13 to 15 amino acids, 15 to 20 amino acids, 21 to 25 amino acids, but preferably 1 to 3 amino acids, 4 to 6 amino acids, 7 to 9 amino acids.

[0350] The study of the insertion site and length of the peptide for enhancing the diversity of the library can be carried out by preparing a molecule into which the peptide is inserted and evaluating the binding of the molecule to CD3. In the evaluation, a method known to those skilled in the art can be appropriately selected, for example, it can be measured by ELISA or FACS (fluorescence activated cell sorting), ALPHA screening (Amplified Luminescent Proximity Homogeneous Assay) or BIACORE method using surface plasmon resonance (SPR) phenomenon.

[0351] As one non-limiting embodiment of the present invention, an antibody library for obtaining antibodies that bind to CD3 and a second antigen can be designed as follows.

[0352] Step 1: Select amino acids that retain CD3 binding ability (CD3 binding is more than 80% of the unmodified antibody)

[0353] For example, a library for obtaining antibodies that bind to CD3 and a second antigen can be prepared by expressing the amino acids selected in step 1.

[0354] As one non-limiting embodiment of the present invention, an antibody library for obtaining antibodies that bind to CD3 and a second antigen can be designed as follows.

[0355] Step 1: Select amino acids that retain CD3 binding ability (CD3 binding is more than 80% of the unmodified antibody)

[0356] Step 2: Inserting amino acids between 99-100 (Kabat numbering) of H chain CDR3 For example, by inserting amino acids into the CDR3 region in step 2 in addition to step 1, a library for obtaining antibodies that bind to CD3 and a second antigen can be prepared with enhanced diversity of the library.

[0357] As a non-limiting embodiment of the present invention, an antibody library for obtaining antibodies that bind to CD3 and a second antigen can be designed as follows.

[0358] Step 1: Select amino acids that retain CD3 binding ability (CD3 binding is more than 80% of the unmodified antibody);

[0359] Step 2: Select amino acids whose ECM binding is within 10 times of MRA binding compared to before the change;

[0360] Step 3: Insert amino acids between 99-100 (Kabat numbering) of CDR3 of the H chain.

[0361] For example, by adding step 2 in addition to steps 1 and 3, amino acids represented in the library can be selected for which ECM binding is not enhanced, but the method is not limited thereto. Furthermore, even if the library is designed without going through step 2, the antigen-binding molecules obtained from the library can be measured for ECM binding and evaluated.

[0362] As a non-limiting embodiment of the present invention, when VH region CE115HA000 (SEQ ID NO: 52) is used as a template sequence for a CD3 (CD3ε) binding antibody, examples of amino acids used for library design include any one or more of the amino acids at positions 11, 31, 52a, 52b, 52c, 53, 54, 56, 57, 61, 72, 78, 98, 99, 100, 100a, 100b, 100c, 100d, 100e, 100f, 100g, and 101 contained in the heavy chain variable region according to the Kabat numbering.

[0363] Preferably, the library with V11L / L78I amino acid changes added to VH region CE115HA000 (SEQ ID NO: 52) is added, but not limited thereto. Furthermore, preferably, the library with V11L / D72A / L78I / D101Q amino acid changes added to VH region CE115HA000 (SEQ ID NO: 52) is added, but not limited thereto.

[0364] As a non-limiting embodiment of the present invention, when the VL region GLS3000 (SEQ ID NO: 53) is used as a template sequence for a CD3 (CD3ε) binding antibody, examples of amino acids used for library design include any one or more of the amino acids at positions 24, 25, 26, 27, 27a, 27b, 27c, 27e, 30, 31, 33, 34, 51, 52, 53, 54, 55, 56, 74, 77, 89, 90, 92, 93, 94, 96, and 107 according to the Kabat numbering system contained in the light chain variable region.

[0365] The designed library in the present invention includes: utilizing 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) to design an antigen-binding domain in which the amino acids at a specific site are changed to the desired amino acids, or a library comprising multiple variants of an antigen-binding molecule containing the antigen-binding domain, but is not particularly limited to this scheme.

[0366] The “one or more amino acids” in the present invention does not particularly limit the number of amino acids, and may be 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 amino acids.

[0367] Regarding the presentation of fusion polypeptides, the fusion polypeptides of the variable regions of antigen-binding molecules can be presented on the surface of cells, viruses or phagemid particles by various schemes. These schemes include single-chain Fv fragments (scFv), F(ab) fragments and multivalent forms of these fragments. The multivalent form is preferably a dimer of ScFv, Fab or F(ab'), which are referred to as (ScFv)2, F(ab)2 and F(ab')2 in this specification. One of the reasons why the presentation of multivalent forms is preferred is that low-affinity clones can usually be identified by presentation in a multivalent form; or clones with multiple antigen binding sites that are rare in the selection process can be more effectively selected.

[0368] The method of presenting a fusion polypeptide containing an antibody fragment on the surface of a phage is well known in the art, for example, as described in WO1992001047 and this specification. In addition, related methods are also described in WO1992020791, WO1993006213, WO1993011236 and 1993019172, and those skilled in the art can use these methods appropriately. In other known documents (HR Hoogenboom & G. Winter (1992) J. Mol. Biol. 227, 381-388, WO1993006213 and WO1993011236), it is shown that the use of artificially rearranged variable region gene repertoires identifies antibodies against various antigens presented on the surface of a phage.

[0369] In the case of constructing a vector for presentation in the scFv scheme, the nucleic acid sequence encoding the light chain variable region and the heavy chain variable region of the antigen binding molecule is contained in the vector. Usually, the nucleic acid sequence encoding the heavy chain variable region of the antigen binding molecule is fused with the viral coat protein constituent. The nucleic acid sequence encoding the light chain variable region of the antigen binding molecule is connected to the heavy chain variable region of the antigen binding molecule through the nucleic acid sequence encoding the peptide linker. The peptide linker usually contains about 5 to 15 amino acids. For example, other sequences of coding tags that are advantageous for purification or detection can be optionally fused to the 3' end of either or both nucleic acid sequences encoding the light chain variable region of the antigen binding molecule or the heavy chain variable region of the antigen binding molecule.

[0370] In the case of constructing a vector for presentation in the F(ab) scheme, the nucleic acid sequence encoding the variable region of the antigen binding molecule and the constant region of the antigen binding molecule is included in the vector. The nucleic acid sequence encoding the light chain variable region is fused with 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 with the nucleic acid sequence encoding the heavy chain constant CH1 region. Usually, the nucleic acid sequence encoding the heavy chain variable region and the constant region is fused with 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 in the form of a fusion with at least a part of the viral coat protein, and the light chain variable region and the constant region are expressed separately with the heavy chain viral coat fusion protein. The mutual binding of the heavy chain and the light chain, the binding can be a covalent bond or a non-covalent bond. For example, other sequences of the coding polypeptide tags that are advantageous for purification or detection can be optionally fused with either or both of the 3' ends of the nucleic acid sequence encoding the light chain constant region of the antigen binding molecule or the 3' ends of the nucleic acid sequence encoding the heavy chain constant region of the antigen binding molecule.

[0371] Regarding the introduction of the vector into the host cell, the vector constructed above is introduced into the host cell for amplification and / or expression. The vector can be introduced into the host cell by known transformation methods including electroporation, calcium phosphate precipitation, etc. In the case where the vector is an infectious particle such as a virus, the vector itself invades the host cell. By transfecting the host cell with a replicable expression vector inserted with a polynucleotide encoding the fusion protein and producing phage particles by known methods, the fusion protein can be presented on the surface of the phage particle.

[0372] Replicable expression vectors can be introduced into host cells using various methods. In a 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 about 6 to 48 hours (or until OD600nm reaches 0.6 to 0.8), and then the culture supernatant is removed by centrifugation of the culture medium (for example, by decantation). In the early stage of purification, it is preferred to resuspend the cell mass in a buffer (for example, 1.0 mM HEPES (pH 7.4)). Next, the supernatant is removed from the suspension by centrifugation again. The obtained cell mass is resuspended in, for example, glycerol diluted to 5-20% V / V. The supernatant is removed from the suspension by centrifugation again to obtain a cell mass. By resuspending the cell mass in water or diluted glycerol, the final cell concentration is adjusted to the desired concentration using water or diluted glycerol according to the measured value of the cell concentration of the obtained suspension.

[0373] For example, as a preferred recipient cell, there can be cited: Escherichia coli strain SS320 that can respond to electroporation (Sidhu et al. (Methods Enzymol. (2000) 328, 333-363)). Escherichia coli strain SS320 is prepared as follows: under sufficient conditions for transferring a fertility episome (F' plasmid) or XL1-BLUE to MC1061 cells, MC1061 cells are mated (crossed) with XL1-BLUE cells. For the Escherichia coli strain SS320 deposited in ATCC (10801 University Boulevard, Manassas, Virginia), its deposit number is 98795. Any F' episome of a replicable phage in this strain can be used in the present invention. Suitable episomes can be obtained from strains deposited with the ATCC or from commercially available products (TG1, CJ236, CSH18, DHF', ER2738, JM101, JM103, JM105, JM107, JM109, JM110, KS1000, XL1-BLUE, 71-18, etc.).

[0374] In electroporation, if a higher DNA concentration (about 10 times) is used, the conversion rate is improved, and the amount of DNA converted to the host cell increases. Using high bacterial concentration also improves efficiency (about 10 times). By increasing the amount of DNA transferred, a larger library with a larger number of independent clones with greater diversity and different sequences can be made. With regard to transformed cells, usually, selection is made by whether to proliferate on a culture medium containing antibiotics.

[0375] Furthermore, the present invention provides a nucleic acid encoding an antigen-binding molecule of the present invention. The nucleic acid of the present invention may be in any form, such as DNA or RNA.

[0376] Furthermore, the present invention provides a vector containing the nucleic acid of the present invention. As for the type of vector, those skilled in the art can appropriately select it according to the host cell into which the vector is to be introduced, and for example, the above-mentioned vectors can be used.

[0377] Furthermore, the present invention relates to a host cell transformed with the vector of the present invention. A person skilled in the art can appropriately select a host cell, and for example, the host cells described above can be used.

[0378] In addition, the present invention provides a kind of pharmaceutical composition, the pharmaceutical composition contains antigen binding molecules of the present invention and medically acceptable carrier.The pharmaceutical composition of the present invention can be introduced into the antigen binding molecules of the present invention and pharmaceutically acceptable carriers according to known methods to prepare preparations.For example, it can be used non-orally in the form of injection of sterile solution or suspension of pharmaceutically acceptable liquid other than water or water.For example, it is possible to appropriately combine pharmacologically acceptable carriers or media, specifically, appropriately combine sterile water or physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, solvent, preservative, adhesive, etc., with the unit dosage form required for the pharmaceutical implementation generally recognized to be mixed, thus preparing preparations. Specifically, as carriers, there can be mentioned: light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylamino acetate, polyvinyl pyrrolidone, gelatin, medium chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, white sugar, carboxymethyl cellulose, corn starch, inorganic salts, etc. The amount of active ingredients in these preparations is set so as to obtain an appropriate volume within the indicated range.

[0379] The sterile composition for injection can be prepared using a solvent such as distilled water for injection according to the usual formulation method. As aqueous solutions for injection, there can be mentioned: for example, physiological saline, isotonic solutions containing glucose or other adjuvants (for example, D-sorbitol, D-mannose, D-mannitol, sodium chloride). It can be used in combination with an appropriate dissolution aid, such as alcohol (specifically, ethanol), polyols (for example, propylene glycol, polyethylene glycol), and nonionic surfactants (for example, polysorbate 80 (TM), HCO-50).

[0380] As the oily liquid, sesame oil and soybean oil can be mentioned, and benzyl benzoate and / or benzyl alcohol can also be used as a dissolving aid. In addition, it can also be matched with a buffer (for example, phosphate buffer and sodium acetate buffer), an analgesic (for example, procaine hydrochloride), a stabilizer (for example, benzyl alcohol and phenol), and an antioxidant. The prepared injection is usually filled in an appropriate ampoule. It is preferably administered non-orally, and specifically, it can be cited: an injection form, a nasal administration form, a pulmonary administration form, a transdermal administration form, etc. As an example of an injection form, for example, it can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.

[0381] In addition, an appropriate administration method can be selected according to the patient's age and symptoms. The dosage of the pharmaceutical composition containing a polypeptide or a polynucleotide encoding a polypeptide can be selected: for example, in the range of 0.0001 mg to 1000 mg / kg body weight / time. Alternatively, the dosage can be selected: for example, in the range of 0.001 to 100000 mg / body per patient, but is not limited to these values. The dosage and administration method vary according to the patient's weight, age, symptoms, etc., and can be appropriately selected by those skilled in the art.

[0382] In addition, the present invention provides: a method for treating cancer, the method comprising the step of administering an antigen-binding molecule of the present invention; an antigen-binding molecule of the present invention for use in the treatment of cancer; use of the antigen-binding molecule of the present invention in the preparation of a therapeutic drug for cancer; and a method for preparing a therapeutic drug for cancer, the method comprising the step of using the antigen-binding molecule of the present invention.

[0383] It should be noted that the correspondence between the three-letter symbols and the single-letter symbols of the amino acids used in this specification is as follows:

[0384] Alanine: Ala: A

[0385] Arginine: Arg: R

[0386] Asparagine: Asn: N

[0387] Aspartic acid: Asp: D

[0388] Cysteine: Cys:C

[0389] Glutamine: Gln: Q

[0390] Glutamate: Glu:E

[0391] Glycine: Gly: G

[0392] Histidine: His:H

[0393] Isoleucine: Ile:I

[0394] Leucine: Leu: L

[0395] Lysine: Lys:K

[0396] Methionine: Met: M

[0397] Phenylalanine: Phe:F

[0398] Proline: Pro:P

[0399] Serine:S

[0400] Threonine: Thr:T

[0401] Tryptophan: Trp: W

[0402] Tyrosine: Tyr: Y

[0403] Valine:Val:V

[0404] It is obvious to those skilled in the art that any combination of one or more embodiments described in this specification is also included in the present invention as long as there is no technical contradiction based on the technical common sense of those skilled in the art.

[0405] It should be noted that all prior art documents cited in this specification are incorporated into this specification as reference.

[0406] The present invention is further illustrated by the following examples, but is not limited thereto.

[0407] Example

[0408] [Example 1] Concept of modified immunoglobulin variable (Fab) region that binds to CD3 (first antigen) and other antigens (second antigen) and does not bind to CD3 (first antigen) and the other antigens (second antigen) on different cells at the same time It is believed that when a cross-linking reaction of activated FcγR occurs due to simultaneous binding of immunoglobulin to two or more active FcγR molecules, or simultaneous binding to other antigens and active FcγR, it is possible to transmit ITAM signal of FcγR and activate immune cells. As described above, since one molecule of IgG type antibody can only bind to one molecule of FcγR, only in the presence of antigen, two or more active FcγR molecules will cross-link and activate immune cells.

[0409] In addition, when an IgG antibody binds to an antigen through the variable region (Fab), it can simultaneously bind to one molecule of FcγR through the Fc region, thereby causing cross-linking between cells expressing the antigen and FcγR-expressing cells. Depending on the cells expressing the antigen, there are also situations where the cross-linking of the antigen and FcγR is not ideal. Specifically, for example, when the antigen is CD3, immune activation such as cytokine release occurs due to cross-linking between T cells and FcγR-expressing cells (J. Immunol. (1999) Aug 1, 163 (3), 1246-52). In such a case, by introducing changes into the Fc region, the binding activity to FcγR is eliminated, and the cross-linking reaction between the antigen and FcγR can be prevented (Advanced Drug Delivery Reviews (2006) 58, 640-656). Similarly, when the antigen of IgG antibodies is TNFR superfamily molecules such as CD40, OX40, CD27, CD3, and TLRs such as TLR2, TLR4, TLR8, and TLR9, when cross-linking occurs via FcγR, immune activation occurs throughout the body, so it is not preferred to simultaneously bind to these molecules expressed in other cells.

[0410] On the other hand, although existing multispecific antibodies bind to multiple antigens at the same time, it is sometimes not preferred to bind to multiple antigens at the same time depending on the combination of antigens. For example, integrin αvβ3, which is known as an adhesion molecule, is expressed in various cancer cells and blood vessels around tumors, and can therefore be used as a target molecule for tumor targeting (R.Haubner, PLoS Med., 2, e70 (2005)), but on the other hand, it is also expressed in various normal cells (Thromb Haemost. 1998 Nov; 80 (5): 726-34.). Therefore, it is believed that if a multispecific antibody binds to both CD3 and integrin αvβ3 at the same time, normal cells may be damaged by the strong cytotoxic activity produced by T cells.

[0411] Therefore, as a method for controlling such undesirable cross-linking reactions, it is considered that there is a variable region (Dual Binding Fab) in which one part of a variable (Fab) region binds to the first antigen and the other part of the variable (Fab) region that does not participate in the binding binds to the second antigen (Fab). Figure 1 ). At this time, if Figure 1 As shown in FIG. 1 , when two adjacent parts in a variable (Fab) region are necessary for binding to each antigen, if the first antigen binds, the binding to the second antigen is inhibited, and similarly, if the second antigen binds, the binding to the first antigen is inhibited. Therefore, the improved antibody having such a dual-binding Fab property cannot bind to the first antigen and the second antigen at the same time, so it is believed that the cross-linking reaction between the first antigen and the second antigen does not occur ( Figure 2 In addition, when the first antigen and the second antigen are not expressed on the cell membrane like soluble proteins, or when both are present on the same cell, they can bind to both the first antigen and the second antigen at the same time, but when they are expressed on different cells, they will not bind to each other at the same time, and the two cells will not cross-link. This situation is also considered to be a dual-binding Fab ( Figure 3 On the other hand, it is believed that the other antigen (third antigen) bound to the variable (Fab) region undergoes a cross-linking reaction with the first antigen ( Figure 4 ), and also cross-links with the second antigen ( Figure 5 ) As the constant region of the antibody, either an Fc region that binds to FcγR or an Fc region whose binding activity to FcγR is reduced can be used.

[0412] When utilizing the properties of such dual-binding Fabs, for example, in the technology of killing cancer cells expressing cancer antigens by redirecting T cells via antibodies, cancer specificity can be further improved by further having an integrin targeting function in cancer tissues.

[0413] That is, when the variable (Fab) region is modified to form a dual-binding Fab, thereby imparting the following properties, a Fab having Figure 1 Antibodies with the indicated effects.

[0414] 1. Having binding activity against the first antigen;

[0415] 2. Has binding activity against the second antigen;

[0416] 3. Does not bind to the first and second antigens at the same time.

[0417] It should be noted that “does not bind to the first antigen and the second antigen at the same time” also includes the following situations: the cells expressing the first antigen and the cells expressing the second antigen do not cross-link, or do not bind to the first antigen and the second antigen expressed on their respective cells at the same time; and the first antigen and the second antigen are not expressed on the cell membrane like soluble proteins, or, when the two are present on the same cell, can bind to the first antigen and the second antigen at the same time, but cannot bind to the two at the same time when they are expressed on different cells.

[0418] Similarly, when the variable (Fab) region is modified to form a dual-binding Fab, the following properties can be conferred, for example, Figure 6 Antibodies with the indicated effects.

[0419] 1. Has binding activity against the first antigen on T cells;

[0420] 2. Has binding activity against the second antigen on antigen presenting cells;

[0421] 3. Does not bind to the first and second antigens at the same time.

[0422] [Example 2] Preparation of Anti-human and Cynomolgus Monkey CD3ε Antibody CE115 (2-1) Preparation of Hybridomas Using Human CD3 and Cynomolgus Monkey CD3-Expressing Cells to Immunize Rats As described below, SD rats (female, 6 years old at the start of immunization, Charles River, Japan) were immunized with Ba / F3 cells expressing human CD3εγ or cynomolgus monkey CD3εγ. The first immunization was defined as day 0, and on day 0, Freund's complete adjuvant (Difco) and 5×10 7 On day 14, Freund's incomplete adjuvant (Difco) and 5×10 7 Cynomolgus monkey CD3εγ-expressing Ba / F3 cells were intraperitoneally administered together, and then 5×10 7 Human or cynomolgus monkey CD3εγ-expressing Ba / F3 cells were administered intraperitoneally alternately for a total of 4 times. One week after the final administration of CD3εγ (day 49), human CD3εγ-expressing Ba / F3 cells were intravenously administered as a booster immunization, and 3 days later, rat spleen cells and mouse myeloma cells SP2 / 0 were fused by a conventional method using PEG1500 (Roche Diagnostics). The fused cells, i.e., hybridomas, were cultured in RPMI1640 medium containing 10% FBS (hereinafter referred to as 10% FBS / RPMI1640).

[0423] On the second day after fusion, (1) the fused cells were suspended in a semi-fluidized culture medium (StemCells) and hybridoma selection and colonization were performed.

[0424] On the 9th or 10th day after fusion, colonies of hybridomas were picked up and one colony was inoculated in each well of a 96-well plate added with HAT selection medium (10% FBS / RPMI1640, 2 vol% HAT 50× concentrate (Dainippon Pharmaceutical), 5 vol% BM-Condimed H1 (Roche Diagnostics)). After culturing for 3 to 4 days, the culture supernatant of each well was recovered and the rat IgG concentration in the culture supernatant was measured. For the culture supernatant in which rat IgG could be confirmed, clones that produced antibodies that specifically bound to human CD3εγ were selected by cell-ELISA using Ba / F3 cells expressing human CD3εγ or Ba / F3 cells that did not express human CD3εγ. Figure 7 ). Then, the cross-reactivity with monkey CD3εγ was evaluated by cell-ELISA with cynomolgus monkey CD3εγ-expressing Ba / F3 cells attached ( Figure 7 ).

[0425] (2-2) Preparation of chimeric antibodies against human and monkey CD3ε

[0426] Total RNA was extracted from hybridoma cells using RNeasy Mini kit (QIAGEN), and cDNA was synthesized using SMART RACE cDNA amplification kit (BD Biosciences). Using the prepared cDNA, the antibody variable region gene was inserted into the cloning vector by PCR. The nucleotide sequence of each DNA fragment was determined according to the method described in the attached manual using the BigDye Terminator cycle sequencing kit (Applied Biosystems) and the DNA sequencer ABI PRISM 3700 DNA Sequencer (Applied Biosystems). The CDR and FR of the CE115 H chain variable region (SEQ ID NO: 13) and the CE115 L chain variable region (SEQ ID NO: 14) were determined according to the Kabat numbering.

[0427] The chimeric antibody H chain gene formed by combining the rat antibody H chain variable region with the human antibody IgG1 chain constant region, and the chimeric antibody L chain gene formed by combining the rat antibody L chain variable region with the human antibody Kappa chain constant region were inserted into an animal cell expression vector. Using the prepared expression vector, the CE115 chimeric antibody was expressed and purified (Reference Example 1).

[0428] (2-3) Preparation of EGFR_ERY22_CE115

[0429] Next, using IgG of anti-cancer antigen (EGFR) as the basic framework, a molecule in which the unilateral Fab was replaced with a binding domain for CD3ε was produced. At this time, as in the above case, the Fc of IgG as the basic framework used a silent Fc with reduced binding to FcgR (Fcγ receptor). As the binding domain for EGFR, cetuximab-VH (SEQ ID NO: 15) and cetuximab-VL (SEQ ID NO: 16) as the variable region of cetuximab were used. As the antibody H chain constant region, G1d obtained by removing Gly and Lys at the C-terminus of IgG1, A5 obtained by introducing D356K and H435R mutations into G1d, and B3 obtained by introducing K439E mutations into G1d were used, and cetuximab-VH-G1d (SEQ ID NO: 17), cetuximab-VH-A5 (SEQ ID NO: 18), and cetuximab-VH-B3 (SEQ ID NO: 19) in combination with cetuximab-VH were prepared according to the method of Reference Example 1. It should be noted that when H1 is used as the name of the antibody H chain constant region, the sequence corresponding to the H chain of the antibody having cetuximab-VH in the variable region is shown as cetuximab-VH-H1.

[0430] Here, when the amino acid change is shown, it is shown as D356K. The first letter (equivalent to D356K) refers to the letter when the amino acid residue before the change is written in a single letter, the subsequent number (equivalent to 356 in D356K) refers to the EU number of the change site, and the last letter (equivalent to K in D356K) refers to the letter when the amino acid residue after the change is written in a single letter.

[0431] EGFR_ERY22_CE115 ( Figure 8 That is, a series of expression vectors into which polynucleotides encoding EGFR ERY22_Hk (SEQ ID NO: 20), EGFR ERY22_L (SEQ ID NO: 21), CE115_ERY22_Hh (SEQ ID NO: 22), and CE115_ERY22_L (SEQ ID NO: 23) were inserted were prepared using methods known to those skilled in the art, such as the PCR method using primers added with appropriate sequences, which is the same as the above method.

[0432] The expression vectors of the following combinations were introduced into FreeStyle293-F cells to transiently express each target molecule.

[0433] Target molecule: EGFR_ERY22_CE115;

[0434] Polypeptides encoded by the polynucleotides inserted into the expression vector: EGFR_ERY22_Hk, EGFR_ERY22_L, CE115_ERY22_Hh, CE115_ERY22_L.

[0435] (2-4) Purification of EGFR_ERY22_CE115

[0436] The obtained culture supernatant was added to an anti-FLAG M2 column (Sigma), the column was washed, and then eluted with 0.1 mg / mL FLAG peptide (Sigma). The component containing the target molecule was added to a HisTrap HP column (GE Healthcare), the column was washed, and then eluted with an imidazole concentration gradient. The component containing the target molecule was concentrated by ultrafiltration, and then the component was added to a Superdex 200 column (GE Healthcare), and only the monomer component of the eluate was recovered, thereby obtaining each purified target molecule.

[0437] (2-5) Cytotoxic activity assay using human peripheral blood mononuclear cells

[0438] (2-5-1) Preparation of human peripheral blood mononuclear cell (PBMC) solution 50 mL of peripheral blood was collected from healthy volunteers (adults) using a syringe pre-injected with 100 μL of 1,000 unit / mL heparin solution (5,000 units of Novo-Heparin for injection, Novo Nordisk). The blood was diluted 2-fold with PBS (-) and divided into 4 equal parts. The obtained peripheral blood was added to a Leucosep lymphocyte separation tube (Cat. No. 227290, Greiner bio-one) pre-injected with 15 mL of Ficoll-Paque PLUS and centrifuged. The separation tube was centrifuged (2,150 rpm, 10 minutes, room temperature) to separate and collect the mononuclear cell component layer. The cells of the mononuclear cell fraction were washed once with Dulbecco's modified Eagle's medium containing 10% FBS (Sigma, hereinafter referred to as 10% FBS / D-MEM), and then the cells were adjusted with 10% FBS / D-MEM to a cell density of 4×10 6 The cell solution thus prepared was used as a human PBMC solution for subsequent experiments.

[0439] (2-5-2) Determination of cytotoxic activity

[0440] The cytotoxic activity was evaluated based on the cell proliferation inhibition rate using the xCELLigence real-time cell analyzer (Roche Diagnostics). The target cells used were the SK-pca13a cell line established by forced expression of human EGFR in the SK-HEP-1 cell line. SK-pca13a was detached from the culture dish and inoculated on an E-Plate 96 plate (Roche Diagnostics) at 100 μL / well to a concentration of 1×10 4 cells / well, and the xCELLigence real-time cell analyzer was used to measure live cells. The next day, the plate was removed from the xCELLigence real-time cell analyzer, and 50 μL of each antibody adjusted to each concentration (0.004, 0.04, 0.4, 4 nM) was added to the plate. The reaction was allowed to proceed at room temperature for 15 minutes, and then 50 μL of the human PBMC solution (2×10 5 Cells / well), the plate was placed again in the xCELLigence real-time cell analyzer to start measuring live cells. The reaction was carried out under 5% carbon dioxide and 37°C, and the cell proliferation inhibition rate (%) was calculated from the Cell Index value 72 hours after adding human PBMC using the following formula. It should be noted that the Cell Index value used in the calculation was standardized by taking the Cell Index value just before adding the antibody as 1.

[0441] Cell proliferation inhibition rate (%) = (AB) × 100 / (A-1)

[0442] A shows the average value of Cell Index values ​​of wells without antibody addition (only target cells and human PBMC), and B shows the average value of Cell Index values ​​of each well. The experiment was performed in triplicate.

[0443] When CE115 was used to measure the cytotoxic activity of EGFR_ERY22_CE115 using PBMC prepared from human blood as effector cells, extremely strong activity was confirmed ( Fig. 9 ).

[0444] [Example 3] Preparation of an Antibody that Binds to CD3 and Human Integrin αvβ3 but Not Both Simultaneously

[0445] like Figures 1 to 6As shown, a dual-binding Fab refers to a molecule that binds to CD3 (the first antigen) and the target antigen (the second antigen) through the variable (Fab) region, but does not bind to CD3 (the first antigen) and the target antigen (the second antigen) at the same time. When amino acid changes are introduced into the Fab region of an antibody that binds to CD3 (the first antigen) in order to bind to the second antigen, amino acid changes are usually introduced into two H chains or two L chains. However, when changes are introduced into two H chains or two L chains, the two Fabs of the antibody bind to two antigens respectively, so that the two Fabs may bind to CD3 (the first antigen) and the target antigen (the second antigen) at the same time and cross-link. Therefore, one Fab of the antibody is used as a Fab that binds to the third antigen or does not bind to any substance, and the other Fab is used as a dual-binding Fab, so that the cross-linking reaction between CD3 (the first antigen) and the target antigen (the second antigen) does not occur.

[0446] (3-1) Preparation of an antibody that binds to CD3 and human integrin αvβ3 but not to both simultaneously Integrin αvβ3, which is known as an adhesion molecule, is expressed in many cancer cells and blood vessels around tumors, and thus can be used as a target molecule for targeting tumors. However, it is also known to be expressed in various normal cells (Thromb Haemost. 1998 Nov; 80(5): 726-34.). Therefore, it is thought that if it binds to CD3 and integrin αvβ3 simultaneously, normal cells may be damaged by the strong cytotoxic activity produced by T cells. Therefore, it is thought that if a molecule that does not bind to CD3 and integrin αvβ3 simultaneously can be prepared, it is possible to target anti-EGFR antibody molecules to tumor cells expressing integrin αvβ3 without causing damage to normal cells. That is, the present invention studied the method of obtaining a dual-binding Fab molecule which binds to EGFR via one variable region (Fab), binds to CD3 as the first antigen via the other variable region, and also binds to integrin αvβ3 as the second antigen, and does not bind to CD3 and integrin αvβ3 simultaneously.

[0447] In other words, if it can be shown that "the molecule binds to CD3 and the Fab region in the absence of integrin αvβ3, the molecule binds to integrin αvβ3 and the Fab region in the absence of CD3, 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", a target bi-binding Fab molecule having the characteristics of bi-binding Fab (i.e., can bind to CD3 and a second antigen, and does not bind to CD3 and the second antigen at the same time) can be developed.

[0448] (3-2) Acquisition of an Antibody Having a Fab Region Binding to Integrin αvβ3

[0449] As a method for obtaining a dual-binding Fab molecule, a method of using a library and a method of inserting a peptide known to have binding activity to a protein are considered. As a peptide having binding activity to integrin αvβ3, RGD (Arg-Gly-Asp) peptide is known. Therefore, according to Reference Example 1, a heterodimeric antibody obtained by inserting an RGD peptide 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) as an antibody binding to CD3ε was prepared, and the heterodimeric antibody used Fab on one side as the EGFR binding domain and Fab on the other side as the CD3 binding domain and the integrin αvβ3 binding domain. That is, 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) and polynucleotides encoding any of the following fragments were inserted simultaneously:

[0450] CE115_2ERY22_Hh (SEQ ID NO: 24, Kabat numbering 52b-53 substituted with K and N, respectively),

[0451] CE115_4ERY22_Hh (SEQ ID NO: 25, Kabat numbering 52b-54 substituted with S and N, respectively),

[0452] CE115_9ERY22_Hh (SEQ ID NO: 26, RGD inserted between Kabat numbering 52a-52b),

[0453] CE115_10ERY22_Hh (SEQ ID NO: 27, RGD inserted between Kabat numbering 52b-52c),

[0454] CE115_12ERY22_Hh (SEQ ID NO: 28, RGD inserted between Kabat numbering 72-73),

[0455] CE115_17ERY22_Hh (SEQ ID NO: 29, Kabat numbering 52b-52c substituted with K and S, respectively),

[0456] CE115_47ERY22_Hh (SEQ ID NO: 30, RGD inserted between Kabat numbering 98-99),

[0457] CE115_48ERY22_Hh (SEQ ID NO: 31, RGD inserted between Kabat numbering 99-100),

[0458] · CE115_49ERY22_Hh (SEQ ID NO: 32, RGD inserted between Kabat numbering 100-100a).

[0459] In addition, as a control, an antibody (EH240-Kn125 / EH240-H1076 / L73; SEQ ID NO: 33 / 34 / 35) in which an 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 that binds to integrin αvβ3 via the CH3 region is considered to be able to bind to CD3 and integrin αvβ3 simultaneously.

[0460] (3-3) Confirmation of the binding of integrin αvβ3 to antibodies

[0461] The binding of the molecule having the RGD (Arg-Gly-Asp) peptide inserted in the Fab region to the integrin αvβ3 was determined by the electrochemiluminescence method (ECL method). Specifically, 25 μL of biotin-anti-human IgG Ab (Southern biotech) diluted with a TBS solution containing 0.1% BSA, 0.1 g / L calcium chloride, and 0.1 g / L magnesium chloride (referred to as a dilution (+) solution) was added to each well of a Nunc-Immuno (tm) MicroWell (tm) 96-well round plate (Nunc), the antibody solution adjusted to 5 μg / mL or 1 μg / mL, and the integrin αvβ3 to which the sulfo-tag was added (R&D Systems) were mixed, and then incubated overnight at 4°C to form an antibody-antigen complex. 150 μL of 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 the streptavidin plate (MSD), and incubated overnight at 4°C. After removing the blocking solution, wash three times with 250 μL of 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 allow biotin-anti-human IgG Ab to bind to the streptavidin plate. After removing the antibody-antigen complex solution, wash three times with TBS (+) solution, 150 μL of READ buffer (MSD) was added to each well, and the luminescent signal of sulfo-tag was detected by Sector Imager 2400 (MSD).

[0462] The results are shown in Fig.11 EGFR ERY22_Hk / EGFR ERY22_L / CE115 ERY22_Hh / CE115_ERY22_L as the parent antibody 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 EGFR ERY22_Hk / EGFR ERY22_L / CE115_47ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_48 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR Binding to integrin αvβ3 was observed in ERY22_L / CE115_49ERY22_Hh / CE115_ERY22_L.

[0463] (3-4) Confirmation of binding between CD3 (CD3ε) and antibodies

[0464] Next, the ECL method was used to determine whether the antibody prepared in the previous item that binds to integrin αvβ3 via the Fab region retains binding activity to CD3. Specifically, 25 μL of biotin-anti-human IgG Ab (Southern biotech) diluted with a TBS solution containing 0.1% BSA (referred to as dilution (-) solution), an antibody solution adjusted to 5 μg / mL or 1 μg / mL, and a CD3ε homodimer protein to which a sulfo-tag was attached were added to each well of a Nunc-Immuno (tm) MicroWell (tm) 96-well circular plate (Nunc), mixed, and then incubated overnight at 4°C to form an antibody-antigen complex. 150 μL of a TBS solution containing 0.5% BSA (referred to as a blocking (-) solution) was added to each well of a streptavidin plate (MSD), and incubated overnight at 4°C. After removing the blocking solution, the plate was washed three times with 250 μL of a TBS solution (-) solution. 75 μL of antibody-antigen complex solution was added to each well and incubated at room temperature for 2 hours to allow biotin-anti-human IgG Ab to bind to the streptavidin plate. After removing the antibody-antigen complex solution, the plate was washed three times with TBS(-) solution, and 150 μL of READ buffer (MSD) was added to each well, and the luminescent signal of sulfo-tag was detected by Sector Imager 2400 (MSD).

[0465] The results are shown in Fig.12In addition to EGFR ERY22_Hk / EGFR ERY22_L / CE115ERY22_Hh / CE115_ERY22_L as the parent antibody, 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, Binding to CD3 was also observed in ERY22_L / CE115_12ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_17 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_47 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_48 ERY22_Hh / CE115_ERY22_L, and EGFR ERY22_Hk / EGFRERY22_L / CE115_49 ERY22_Hh / CE115_ERY22_L.

[0466] (3-5) Confirmation by ECL method that integrin αvβ3 and CD3 do not bind to the Fab region simultaneously

[0467] From the results obtained so far, a molecule having binding activity to integrin αvβ3 and also having binding activity to CD3 was obtained. Next, it was determined whether the Fab region prepared so far binds to both CD3 (CD3ε) and integrin αvβ3.

[0468] In the case where a molecule having an RGD (Arg-Gly-Asp) peptide inserted into the Fab region binds to both integrin αvβ3 and CD3, if integrin αvβ3 and CD3 to which biotin is attached are added to the antibody solution, the integrin αvβ3 and CD3 to which biotin is attached bind to both antigens, and thus detection can be performed by the ECL method. Specifically, 25 μL of human CD3ε homodimer protein to which biotin is attached diluted with a diluent (+) solution, an antibody solution adjusted to 10 μg / mL or 5 μg / mL, and integrin αvβ3 to which sulfo-tag is attached (R&D Systems) are added to each well of a Nunc-Immuno (tm) MicroWell (tm) 96-well round plate (Nunc), mixed, and then incubated overnight at 4°C to form an antibody-antigen complex. 150 μL of blocking (+) solution is added to each well of a streptavidin plate (MSD), and incubated overnight at 4°C. After removing the blocking solution, wash 3 times with 250 μL of TBS(+) solution containing 0.1 g / L calcium chloride and 0.1 g / L magnesium chloride. Add 75 μL of antibody-antigen complex solution to each well and incubate at room temperature for 2 hours to allow biotin-anti-human IgG Ab to bind to the streptavidin plate. After removing the antibody-antigen complex solution, wash 3 times with TBS(+) solution, add 150 μL of READ buffer (MSD) to each well, and detect the luminescent signal of sulfo-tag by Sector Imager 2400 (MSD).

[0469] The results are shown in Fig.13 , Fig.14 EGFR ERY22_Hk / EGFR ERY22_L / CE115_2 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_12ERY22_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, thereby detecting strong signals in the ECL assay. On the other hand, in EGFR ERY22_Hk / EGFR ERY22_L / CE115_9 ERY22_Hh / CE115_ERY22_L and EGFR ERY22_Hk / EGFR ERY22_L / CE115_48 ERY22_Hh / CE115_ERY22_L, the signal was weak ( Fig.13In addition, almost no signal was detected in ECL assay for EGFR ERY22_Hk / EGFR ERY22_L / CE115_4ERY22_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_49ERY22_Hh / CE115_ERY22_L ( Fig.14 ). That is, the results suggest that if these antibodies bind to CD3, they do not bind to integrin αvβ3.

[0470] (3-6) Using ECL method to investigate whether integrin αvβ3 and CD3 bind to Fab region simultaneously

[0471] According to the above results, an antibody having the characteristics of a dual-binding Fab molecule that binds to CD3 (CD3ε) and integrin αvβ3 respectively through one Fab, and does not bind to CD3 (CD3ε) and integrin αvβ3 at the same time, can be developed. In this embodiment, for an antibody having a variable region that binds to CD3 as the first antigen, by inserting an RGD peptide that binds to integrin αvβ3 as the second antigen in the variable region into the Fab, a molecule that is endowed with binding activity to the second antigen and does not bind to CD3 and the second antigen at the same time can be obtained. In the same way, by inserting a peptide having binding activity to a protein as exemplified in WO2006036834 into a loop in the Fab, a dual-binding Fab molecule having binding activity to an arbitrary second antigen can be obtained. In addition, a peptide that exhibits binding activity to a protein can be obtained by preparing a peptide library using a method known to those skilled in the art and selecting a peptide having the desired activity (Pasqualini R., Nature, 1996, 380 (6572): 364-6). Furthermore, it is believed that a dual-binding Fab molecule having binding activity to an arbitrary second antigen can be developed by using a library of antigen-binding molecules in which the loops in Fab are lengthened (elongated) as described in Example 5. Since the variable region against the first antigen can be obtained by various methods known to those skilled in the art, it is believed that by using such a library, a dual-binding Fab molecule having binding activity to an arbitrary first antigen and an arbitrary second antigen and being unable to bind to the first antigen and the second antigen simultaneously can be developed.

[0472] The above results show 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 / EGFRERY22_L / CE115_49 ERY22_Hh / CE115_ERY22_L bind to CD3 and integrin αvβ3, but not simultaneously. That is, it was revealed 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 are molecules having dual-binding Fabs, and such molecules can be developed.

[0473] [Example 4] Preparation of an Antibody that Binds to CD3 and Human Toll-like Receptor 2 (TLR2) but Not Both Simultaneously

[0474] (4-1) Preparation of an Antibody that Binds to CD3 and Human TLR2, but Not Both Simultaneously

[0475] TLR2, which is known as a pattern recognition receptor, is mainly expressed in immune cells such as macrophages, dendritic cells or B cells, and can be used as a target molecule for activating immune cells. In addition, it is known that TLR2 is also expressed in normal cells other than immune cells such as epithelial cells or endothelial cells. By combining cancer antigens and CD3 at the same time, T cells expressing CD3 in the tumor environment are recruited, and cancer cells are killed with T cells, but it is believed that by combining cancer antigens and TLR2 at the same time, immune cells expressing TLR2 in the tumor environment can also be recruited and activated. The immune cells recruited by TLR2 take in cancer cells killed by T cells, process the antigens and present them to HLA, so that T cells can be activated, so while activating T cells more effectively, it is also possible to induce acquired immunity. However, it is believed that if CD3 and TLR2 are combined at the same time, it is possible to cause immune cells and normal cells to be damaged by the strong cytotoxic activity produced by T cells. Therefore, it is believed that if a molecule that is not combined with CD3 and TLR2 at the same time can be made, immune cells and normal cells expressing TLR2 can be recruited without causing damage to these cells. That is, the study investigated obtaining a dual-binding Fab molecule that binds to EGFR via one variable region (Fab), binds to CD3 as the first antigen via the other variable region, and also binds to TLR2 as the second antigen, and does not bind to CD3 and TLR2 simultaneously.

[0476] In other words, if it can be shown that "the molecule binds to CD3 and the Fab region in the absence of TLR2, and to TLR2 and the Fab region in the absence of CD3, the molecule that binds to CD3 does not bind to TLR2, or the molecule that binds to TLR2 does not bind to CD3", it is possible to develop a target dual-binding Fab molecule having the characteristics of dual-binding Fab (i.e., it can bind to CD3 and the second antigen, and it does not bind to CD3 and the second antigen at the same time).

[0477] (4-2) Acquisition of Antibodies Having Fab Regions Binding to TLR2

[0478] As a peptide having binding activity to human TLR2, the RWGYHLRDRKYKGVRSHKGVPR peptide (SEQ ID NO: 36) is known. Therefore, according to Reference Example 1, a heterodimeric antibody was prepared in which a TRL2 binding peptide was inserted into the loop portion of the heavy chain of CE115 (heavy chain variable region is SEQ ID NO: 13, light chain variable region is SEQ ID NO: 14), which is an antibody binding to CD3ε, and the heterodimeric antibody used Fab on one side as the EGFR binding domain and Fab on the other side as the CD3 binding domain and TLR2 binding domain. That is, 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) and polynucleotides encoding any of the following fragments were inserted simultaneously:

[0479] CE115_DU21 ERY22_Hh (SEQ ID NO: 37, TRL2 binding peptide inserted between Kabat numbering 52b-52c),

[0480] CE115_DU22 ERY22_Hh (SEQ ID NO: 38, TRL2 binding peptide inserted between Kabat numbering 52b-52c),

[0481] CE115_DU26 ERY22_Hh (SEQ ID NO: 39, TRL2 binding peptide inserted between Kabat numbers 72-73),

[0482] · CE115_DU27 ERY22_Hh (SEQ ID NO: 40, TRL2 binding peptide inserted between Kabat numbers 72-73).

[0483] In addition, as controls, an antibody (CE115_ERY22_DU42_Hh, SEQ ID NO: 41) having a TLR2-binding peptide added to the C-terminus of the CH3 region and an antibody (CE115_ERY22_DU43_Hh, SEQ ID NO: 42) having a peptide having Cys residues at both ends of the TLR2-binding peptide added to the C-terminus of the CH3 region were prepared according to Reference Example 1. It is believed that this molecule that binds to TLR2 via the CH3 region can bind to CD3 and TLR2 simultaneously.

[0484] (4-3) Confirmation of TLR2-antibody binding

[0485] The electrochemiluminescence method (ECL method) was used to determine whether the molecule with a TLR2 binding peptide inserted in the Fab region binds to TLR2. Specifically, 25 μL of biotin-anti-human IgG Ab (Southernbiotech) diluted with a TBS solution containing 0.1% BSA (referred to as a dilution (-) solution) was added to each well of a Nunc-Immuno (tm) MicroWell (tm) 96-well circular plate (Nunc), an antibody solution adjusted to 5 μg / mL or 1 μg / mL, and TLR2 (abnova) attached with a sulfo-tag were mixed, and then incubated overnight at 4°C to form an antibody-antigen complex. 150 μL of a TBS solution containing 0.5% BSA (referred to as a blocking (-) solution) was added to each well of a streptavidin plate (MSD), and incubated overnight at 4°C. After removing the blocking solution, wash three times with 250 μL of a TBS (-) solution. 75 μL of antibody-antigen complex solution was added to each well and incubated at room temperature for 2 hours to allow biotin-anti-human IgG Ab to bind to the streptavidin plate. After removing the antibody-antigen complex solution, the plate was washed three times with TBS(-) solution, and 150 μL of READ buffer (MSD) was added to each well, and the luminescent signal of sulfo-tag was detected by Sector Imager 2400 (MSD).

[0486] The results are shown in Fig.15 EGFR ERY22_Hk / EGFR ERY22_L / CE115 ERY22_Hh / CE115_ERY22_L as the parent antibody showed no binding activity to TLR2. On the contrary, 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 Binding to TLR2 was observed in both ERY22_Hh and CE115_ERY22_L.

[0487] (4-4) Confirmation of binding between CD3 (CD3ε) and antibodies

[0488] Next, the ECL method was used to determine whether the antibody that binds to TLR2 via the Fab region prepared in the previous item retains binding activity to CD3 (CD3ε). Specifically, 25 μL of biotin-anti-human IgG Ab (Southern biotech) diluted with a TBS solution containing 0.1% BSA (referred to as dilution (-) solution) was added to each well of a Nunc-Immuno (tm) MicroWell (tm) 96-well circular plate (Nunc), an antibody solution adjusted to 5 μg / mL or 1 μg / mL, and a CD3ε homodimer protein with a sulfo-tag were mixed, and then incubated overnight at 4°C to form an antibody-antigen complex. 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 plate was washed three times with 250 μL of a TBS solution (-) solution. 75 μL of antibody-antigen complex solution was added to each well and incubated at room temperature for 2 hours to allow biotin-anti-human IgG Ab to bind to the streptavidin plate. After removing the antibody-antigen complex solution, the plate was washed three times with TBS(-) solution, and 150 μL of READ buffer (MSD) was added to each well, and the luminescent signal of sulfo-tag was detected by Sector Imager 2400 (MSD).

[0489] The results are shown in Fig.16 In addition to EGFR ERY22_Hk / EGFR ERY22_L / CE115ERY22_Hh / CE115_ERY22_L as the parent antibody, 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 Binding to CD3 was also observed in ERY22_Hh / CE115_ERY22_L.

[0490] (4-5) Confirmation by ECL method that TLR2 and CD3 do not bind to the Fab region at the same time

[0491] Based on the results obtained so far, a molecule having binding activity to TLR2 and CD3 was obtained. Next, it was determined whether the Fab regions prepared so far bind to both CD3 and TLR2.

[0492] In the case where a molecule having a TLR2 binding peptide inserted into the Fab region binds to both TLR2 and CD3, if TLR2 and CD3 attached with biotin are added to the antibody solution, the molecules will bind to both antigens, and thus can be detected by the ECL method. Specifically, 25 μL of human CD3ε homodimer protein attached with biotin diluted with a diluent (-) solution, an antibody solution adjusted to 10 μg / mL or 5 μg / mL, and TLR2 attached with a sulfo-tag (R&D Systems) were added to each well of a Nunc-Immuno(tm) Micro Well(tm) 96-well round plate (Nunc), mixed, and then incubated overnight at 4°C to form an antibody-antigen complex. 150 μL of blocking (-) solution was added to each well of a streptavidin plate (MSD), and incubated overnight at 4°C. After removing the blocking solution, wash 3 times with 250 μL of TBS(-) solution containing 0.1 g / L calcium chloride and 0.1 g / L magnesium chloride. Add 75 μL of antibody-antigen complex solution to each well and incubate at room temperature for 2 hours to allow biotin-anti-human IgG Ab to bind to the streptavidin plate. After removing the antibody-antigen complex solution, wash 3 times with TBS(-) solution, add 150 μL of READ buffer (MSD) to each well, and detect the luminescent signal of sulfo-tag by Sector Imager 2400 (MSD).

[0493] The results are shown in Fig.17Since EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU42 ERY22_Hh / CE115_ERY22_L and EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU43 ERY22_Hh / CE115_ERY22_L to which a TLR2-binding peptide was added to the CH3 region simultaneously bound to TLR2 and CD3, strong signals were detected in the ECL assay. On the other hand, almost no signal was detected in ECL assay for EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU21 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU22 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU26 ERY22_Hh / CE115_ERY22_L, and EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU27 ERY22_Hh / CE115_ERY22_L. In other words, the results suggest that these antibodies do not bind to TLR2 when they bind to CD3.

[0494] (4-6) Using ECL method to investigate the difference between TLR2 and CD3 binding to Fab region at the same time

[0495] From the above results, it can be seen that: an antibody having the characteristics of a dual-binding Fab molecule that binds to CD3 and TLR2 respectively through one Fab and does not bind to CD3 and TLR2 at the same time can be developed. In this embodiment, for an antibody having a variable region that binds to CD3 as the first antigen, by inserting a RWGYHLRDRKYKGVRSHKGVPR peptide that binds to TLR2 as the second antigen in the variable region into the Fab, a molecule that is endowed with binding activity to the second antigen and does not bind to CD3 and the second antigen at the same time can be obtained. By the same method, by inserting a peptide having binding activity to a protein as exemplified in WO2006036834 into a loop in the Fab, a dual-binding Fab molecule having binding activity to any second antigen can be obtained. In addition, a peptide that shows binding activity to a protein can be obtained by preparing a peptide library using a method known to those skilled in the art and selecting a peptide having the desired activity (Pasqualini R., Nature, 1996, 380 (6572): 364-6)). Furthermore, it is believed that a dual-binding Fab molecule having binding activity to an arbitrary second antigen can be developed by using a library of antigen-binding molecules in which the loops in Fab are lengthened (elongated) as described in Example 5. The variable region against the first antigen can be obtained by various methods known to those skilled in the art, so it can be said that by using such a library, a dual-binding Fab molecule having binding activity to an arbitrary first antigen and an arbitrary second antigen and being unable to bind to the first antigen and the second antigen simultaneously can be developed.

[0496] The above results show 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 / EGFRERY22_L / CE115_DU27 ERY22_Hh / CE115_ERY22_L bind to CD3 and TLR2, but do not bind to CD3 and TLR2 at the same time. That is, it was revealed 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 are molecules having dual-binding Fabs, and such molecules can be developed.

[0497] [Example 5] Antibody modification for producing antibodies that bind to CD3 and a second antigen

[0498] (5-1) Study on the insertion site and length of the peptide capable of binding to the second antigen

[0499] The invention has been studied to obtain a dual-binding Fab molecule that binds to a cancer antigen through one variable region (Fab), binds to CD3 as a first antigen through the other variable region, and also binds to a second antigen, and does not bind to CD3 and the second antigen simultaneously. A heterodimeric antibody in which a GGS peptide is inserted into the loop portion of the heavy chain of CE115, an antibody that binds to CD3ε, was prepared according to Reference Example 1, and the heterodimeric antibody used the Fab on one side as an EGFR binding domain and the Fab on the other side as a CD3 binding domain.

[0500] Specifically, EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE31 ERY22_Hh / CE115_ERY22_L (SEQ ID NOs: 20 / 21 / 43 / 23) in which GGS was inserted between K52B and S52c in CDR2, EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE32 ERY22_Hh / CE115_ERY22_L (SEQ ID NOs: 20 / 21 / 44 / 23) in which a GGSGGS peptide (SEQ ID NO: 90) was inserted, and EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE33 ERY22_Hh / CE115_ERY22_L (SEQ ID NOs: 20 / 21 / 44 / 23) in which a GGSGGSGGS peptide (SEQ ID NO: 91) was inserted were prepared. Similarly, EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE34 ERY22_Hh / CE115_ERY22_L (SEQ ID NOs: 20 / 21 / 46 / 23) in which GGS was inserted between D72 and D73, the loop site in framework 3, EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE35 ERY22_Hh / CE115_ERY22_L (SEQ ID NOs: 20 / 21 / 47 / 23) in which the GGSGGS peptide (SEQ ID NO: 90) was inserted, and EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE36 ERY22_Hh / CE115_ERY22_L (SEQ ID NOs: 20 / 21 / 48 / 23) in which the GGSGGSGGS peptide (SEQ ID NO: 91) was inserted were prepared. ERY22_Hh / CE115_ERY22_L ((SEQ ID NO: 20 / 21 / 48 / 23). In addition, EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE37 ERY22_Hh / CE115_ERY22_L ((SEQ ID NO: 20 / 21 / 49 / 23) in which GGS was inserted between A99 and Y100 in CDR3, EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE38 ERY22_Hh / CE115_ERY22_L ((SEQ ID NO: 20 / 21 / 50 / 23) in which a GGSGGS peptide was inserted, and EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE39 in which a GGSGGSGGS peptide was inserted were prepared. ERY22_Hh / CE115_ERY22_L((SEQ ID NO:20 / 21 / 51 / 23).

[0501] (5-2) Confirmation of the binding of CE115 antibody with GGS peptide inserted into it to CD3ε

[0502] Whether the various antibodies produced maintained binding properties to CD3ε was confirmed by Biacore T100. The biotinylated CD3ε epitope peptide was bound to a CM5 chip via streptavidin, and the produced antibodies were passed as analytes to analyze binding affinity.

[0503] The results are shown in Table 2. The binding affinity of CE35, CE36, CE37, CE38, and CE39 to CD3ε was equivalent to that of CE115, which is the parent antibody. This shows that a peptide that binds to the second antigen can be inserted into these loops. In addition, even in CE36 or CE39 where GGSGGSGGS was inserted, the binding affinity did not decrease, which shows that the insertion of a peptide of at least 9 amino acids at these sites does not affect the CD3ε binding.

[0504] [Table 2]

[0505]

[0506] That is, the results showed that by using such a peptide-inserted CE115 to obtain an antibody that binds to the second antigen, an antibody that can bind to CD3 and the second antigen but not simultaneously can be prepared.

[0507] Here, the amino acid sequence of the peptide to be inserted or substituted is randomly changed according to a well-known method such as site-specific mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) or overlapping extension PCR, and the binding activity of each variant is compared according to the above method to determine the insertion or substitution site or the type and length of its amino acids that can show the target activity even if the amino acid sequence is changed, so that a library can be prepared.

[0508] [Example 6] Library design for obtaining antibodies binding to CD3 and a second antigen

[0509] (6-1) Regarding the antibody library for obtaining antibodies that bind to CD3 and a second antigen (also called a dual Fab library), CD3 (CD3ε) is selected as the first antigen. As methods for obtaining antibodies that bind to CD3 (CD3ε) and any second antigen, the following six methods can be exemplified.

[0510] Method 1: inserting a peptide or polypeptide binding to the second antigen into the Fab domain binding to the first antigen (in this method, in addition to the peptide insertion shown in Examples 3 and 4, G-CSF is also inserted as exemplified in Angew Chem Int Ed Engl. 2013 Aug 5; 52(32): 8295-8), wherein the peptide or polypeptide to be bound can be obtained from a library presenting peptides or polypeptides, but the whole or part of a naturally occurring protein can also be used;

[0511] Method 2: Prepare an antibody library in which various amino acids are presented at positions that can lengthen (extend) the loop in Fab as shown in Example 5, and obtain Fab having binding activity to an arbitrary second antigen from the antibody library using the binding activity to the antigen as an index;

[0512] Method 3: Using an antibody previously prepared by site-specific mutagenesis from a Fab domain known to bind to CD3, the amino acids that maintain binding activity to CD3 are identified, and using the binding activity to the antigen as an indicator, Fabs having binding activity to an arbitrary second antigen are obtained from an antibody library that presents the identified amino acids;

[0513] Method 4: In method 3, an antibody library is further prepared in which various amino acids are presented at positions that can lengthen (extend) the loop in Fab, and Fab having binding activity to an arbitrary second antigen is obtained from the antibody library using the binding activity to the antigen as an index;

[0514] Method 5: In methods 1, 2, 3, and 4, a sugar chain addition sequence (e.g., NxS, NxT, wherein x is an amino acid other than P) is presented, and a sugar chain recognized by a sugar chain receptor is added (e.g., a high mannose type sugar chain is added and recognized by the high mannose receptor. It is known that a high mannose type sugar chain can be obtained by adding kifnosine during antibody expression (MAbs. 2012 Jul-Aug; 4(4): 475-87));

[0515] Method 6: In methods 1, 2, 3, and 4, Cys, Lys, or non-natural amino acids are inserted or substituted at the loop site or at the site where various amino acids can be changed, and a domain (polypeptide or sugar chain, nucleic acid represented by TLR agonist) that binds to the second antigen is added by covalent bond (a method represented by antibody-drug conjugates, a method of binding to Cys, Lys, or non-natural amino acids by covalent bonds, described in mAbs 6: 1, 34-45; January / February 2014, WO2009 / 134891A2, Bioconjug Chem. 2014Feb 19; 25(2): 351-61);

[0516] The above method is used to obtain a dual-binding Fab that binds to the first antigen and the second antigen but does not bind to each other at the same time, and then it is combined with a domain that binds to an arbitrary third antigen (referred to as the other variable region, described in Example 1) by methods known to those skilled in the art, such as common L chain, Cross mab, and Fab arm exchange.

[0517] (6-2) Preparation of an amino acid-modified antibody of a CD3 (CD3ε) binding antibody by site-specific mutagenesis As the template sequence of the CD3 (CD3ε) binding antibody, CE115HA000 (SEQ ID NO: 52) was selected for the VH region, and GLS3000 (SEQ ID NO: 53) was selected for the VL region. Amino acid changes were made at the sites believed to be involved in antigen binding according to Reference Example 1. In addition, pE22Hh (a sequence obtained by adding L234A, L235A, N297A, D356C, T366S, L368A, and Y407V to the sequence after CH1 of natural IgG1, deleting the GK sequence at the C-terminus, and adding the DYKDDDDK sequence (SEQ ID NO: 89); SEQ ID NO: 54) was used for the H chain constant region, and a Kappa chain (SEQ ID NO: 55) was used for the L chain constant region. The sites that were changed are shown in Table 3. In order to evaluate the CD3 (CD3ε) binding activity, an amino acid-modified antibody was obtained as a one-armed antibody (an antibody in which one Fab domain is deleted in natural IgG). Specifically, when the H chain was modified, a chain formed by linking the modified H chain to the constant region pE22Hh and Kn010G3 (a sequence obtained by adding C220S, Y349C, T366W, and H435R to the amino acid sequence after position 216 of natural IgG1, SEQ ID NO: 56) were used as the H chain, and GLS3000 with a kappa chain linked to the 3' side was used as the L chain; when the L chain was modified, a sequence with a kappa chain linked to the 3' side of the modified L chain was used as the L chain, and CE115HA000 and Kn010G3 with pE22Hh linked to the 3' side were used as the H chain, and the expression and purification were performed in FreeStyle293 cells (using the method of Reference Example 1).

[0518] [Table 3]

[0519]

[0520] (6-3) Evaluation of the binding of an amino acid-modified antibody to CD3

[0521] The amino acid variant constructed, expressed and purified in (6-2) was evaluated using Biacore T200 (GE Healthcare). After fixing an appropriate amount of CD3ε homodimer protein on the sensor chip CM4 (GE Healthcare) by the amine coupling method, an appropriate concentration of antibody was injected as the analyte to allow it to interact with the CD3ε homodimer protein on the sensor chip. Thereafter, 10 mmol / L of glycine-HCl (pH 1.5) was injected to regenerate the sensor chip. The measurement was performed at 25°C, and HBS-EP+ (GE Healthcare) was used as the running buffer. Based on the results of the measurement, the dissociation constant K was calculated using a single-cycle kinetic model (1:1 binding RI = 0) for the binding amount and the sensorgram obtained by the measurement. D (M) Biacore T200 evaluation software (GE Healthcare) was used for the calculation of each parameter.

[0522] (6-3-1) Changes in H chain

[0523] The results of the ratio of the binding amount of various H chain variants to CE115HA000 as the antibody before modification are shown in Table 4. That is, when the binding amount of the antibody containing CE115HA000 is X and the binding amount of the H chain one amino acid variant is Y, Z (ratio of binding amount) = Y / X. In this case, Fig.18 As shown in Figure 2, when Z is less than 0.8, the sensorgram shows that the amount of binding is very small, suggesting that the dissociation constant K may not be calculated correctly. D (M). Next, for CE115HA000, the dissociation constants K of various H chain variants were D The ratio of (M) (=KD value of CE115HA000 / KD value of the variant) is shown in Table 5.

[0524] When Z shown in Table 4 is 0.8 or more, it is considered that the binding to CE115HA000, which is the antibody before modification, is maintained, and therefore the antibody library designed to present these amino acids can be used as a Dual Fab library.

[0525] [Table 4]

[0526]

[0527] [Table 5]

[0528]

[0529] (6-3-2) L chain modification The results of the ratio of the binding amount of various L chain variants relative to GLS3000, which is the antibody before modification, are shown in Table 6. That is, when the binding amount of the antibody containing GLS3000 is X and the binding amount of the L chain one amino acid variant is Y, Z (ratio of binding amount) = Y / X. In this case, Fig.18 As shown in Figure 2, when Z is less than 0.8, the sensorgram shows that the amount of binding is very small, suggesting that the dissociation constant K may not be calculated correctly. D (M). Next, the dissociation constants K of various L chain variants relative to GLS3000 were D The ratio of (M) is shown in Table 7.

[0530] When Z shown in Table 6 is 0.8 or more, it is considered that the binding to GLS3000, which is the antibody before modification, is maintained, and therefore the antibody library designed to present these amino acids can be used as a Dual Fab library.

[0531] [Table 6]

[0532]

[0533] [Table 7]

[0534]

[0535] (6-4) Evaluation of the binding of an amino acid-modified antibody to ECM (Extracellular matrix)

[0536] ECM (Extracellular matrix) is one of the components outside cells and exists in various parts of the body. Therefore, it is known that antibodies that strongly bind to ECM have poor blood dynamics (shortened half-life) (WO2012093704A1). Therefore, it is also preferred to select amino acids that do not enhance binding to ECM for the amino acids presented in the antibody library.

[0537] For each H chain or L chain variant, antibodies were obtained according to the method shown in (6-2). Next, ECM binding was evaluated according to the method of Reference Example 2. The values ​​obtained by dividing the ECM binding value (ECL response; ECL response value) of each variant 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 implementation day 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 confirmed in several modifications.

[0538] Among the values ​​shown in Table 8 (H chain) and Table 9 (L chain), considering the effect of enhancing ECM binding caused by multiple changes, the highest 10-fold was adopted as the effective value in the Dual Fab library.

[0539] [Table 8]

[0540]

[0541] [Table 9]

[0542]

[0543] (6-5) Study on the insertion site and length of peptides for enhancing library diversity

[0544] In Example 5, it is shown that the peptide can be inserted into each site using the GGS sequence without losing the binding to CD3 (CD3ε). It is believed that in the Dual Fab library, if the loop can be extended, a library containing more molecules (also expressed as large diversity) will be formed, and Fab domains that bind to various second antigens can be obtained. Therefore, it is assumed that the binding activity will decrease with the insertion of the peptide, so V11L / D72A / L78I / D101Q is added to the CE115HA000 sequence in a manner that the binding activity to CD3ε becomes higher and pE22Hh is connected to obtain a sequence. As in Example 5, a molecule in which a GGS linker is inserted into the obtained sequence is prepared, and the binding of the molecule to CD3 is evaluated. The GGS sequence is inserted between Kabat numbering 99-100. The antibody molecule is expressed in the form of a one-arm antibody. Specifically, the expression and purification were carried out according to Reference Example 1 using the above H chain containing a GGS linker and Kn010G3 (SEQ ID NO: 56) as the H chain and GLS3000 (SEQ ID NO: 53) linked to a Kappa sequence (SEQ ID NO: 55) as the L chain.

[0545] (6-6) Confirmation of the binding of CE115 antibody with GGS peptide inserted into it to CD3

[0546] According to the method described in Example 6, the binding of the modified antibody with the GGS peptide inserted to CD3ε was performed using Biacore. As shown in Table 10, it was clearly shown that the GGS linker can be inserted into the loop site. In particular, the GGS linker can be inserted into the H chain CDR3 region, which is important for antigen binding, and the binding to CD3ε can be maintained even if any 3, 6 or 9 amino acids are inserted. In this study, the GGS linker was used for research, but it is considered that the antibody library can also present various amino acids instead of GGS.

[0547] [Table 10]

[0548] Insert amino acid sequence (99-100) CD3_KD[M] GGS 6.31E-08 GGSGGS (SEQ ID NO: 90) 3.46E-08 GGSGGS (SEQ ID NO: 90) 3.105E-08 GGSGGGS (SEQ ID NO: 92) 4.352E-08 GGSGGGS (SEQ ID NO: 92) 3.429E-08 GGGSGGGS (SEQ ID NO: 93) 4.129E-08 GGGSGGGS (SEQ ID NO: 93) 3.753E-08 GGSGGSGGS (SEQ ID NO: 91) 4.39E-08 GGSGGSGGS (SEQ ID NO: 91) 3.537E-08 No insertion 6.961E-09 CE115HA000 1.097E-07

[0549] (6-7) Using NNS nucleotides to study the insertion of libraries into H chain CDR3

[0550] (6-6) shows that 3, 6, and 9 amino acids can be inserted using a GGS linker. It is believed that antibodies that bind to the second antigen can be obtained by preparing a library with 3, 6, and 9 amino acids inserted and using an antibody acquisition method represented by a conventional phage display method. Therefore, it was studied whether binding to CD3 can be maintained even when 6 amino acids are inserted into CDR3 using NNS nucleotides (presenting various amino acids) and various amino acids are presented at the site where 6 amino acids are inserted. Because it is expected that the binding activity will decrease, primers were designed using NNS nucleotides to insert 6 amino acids between 99-100 (Kabat numbering) in CDR3 of the CE115HA340 sequence (SEQ ID NO: 59) with higher CD3ε binding activity than CE115HA000. The antibody molecule was expressed in the form of a one-armed antibody. Specifically, the H chain and Kn010G3 (SEQID NO: 56) containing the above-mentioned modification were used as the H chain, and GLS3000 (SEQ ID NO: 53) connected with a Kappa sequence (SEQ ID NO: 55) was used as the L chain, and expression and purification were performed according to Reference Example 1. The obtained modified antibody was evaluated for binding according to the method described in (6-3). The results are shown in Table 11. It was clarified that even when various amino acids are present at the site of the extended amino acid, the binding to CD3 (CD3ε) can be maintained. Whether the non-specific binding is enhanced was evaluated by the method shown in Reference Example 2, and the results are shown in Table 12. As a result, when many amino acids with positive side chains are contained in the extended loop of CDR3, the binding to ECM is enhanced, so it is desirable that amino acids with more than three positive side chains are not present in the loop.

[0551] [Table 11]

[0552]

[0553] [Table 12]

[0554]

[0555] (6-7) Design and construction of dual Fab library

[0556] Based on the studies described in Example 6, an antibody library (Dual Fab library) for obtaining antibodies that bind to CD3 and a second antigen was designed as follows.

[0557] Step 1: Select amino acids that retain CD3 (CD3ε) binding ability (CD3 binding capacity is more than 80% of CE115HA000);

[0558] Step 2: Select amino acids whose ECM binding is within 10 times of MRA binding compared to before the change;

[0559] Step 3: Insert 6 amino acids between 99-100 (Kabat numbering) of CDR3 of the H chain.

[0560] It should be noted that since the antigen-binding sites of Fab can be diversified only through step 1, a library for identifying antigen-binding molecules that bind to the second antigen can be obtained. In addition, since the antigen-binding sites of Fab can be diversified only through steps 1 and 3, a library for identifying antigen-binding molecules that bind to the second antigen can be obtained. Even if the library design is not performed through step 2, the ECM binding of the obtained molecules can be measured and evaluated.

[0561] From the above results, it can be seen that in the H chain of the Dual Fab library, the sequence obtained by adding V11L / L78I mutations to the FR (framework) of CE115HA000 was diversified as CDR as shown in Table 13, and the CDR of GLS3000 was diversified in the L chain as shown in Table 14. These antibody library fragments can be synthesized by a DNA synthesis method known to those skilled in the art. As a Dual Fab library, the following libraries can be prepared: (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 L chain with enhanced CD3ε binding described in Example 6; (2) a library in which the H chain is fixed to the original sequence (CE115HA000) or the H chain with enhanced CD3ε binding described in Example 6 and the L chain is diversified as shown in Table 14; (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 library sequence obtained by adding V11L / L78I mutation to the FR (framework) of CE115HA000 and diversifying CDR as shown in Table 13 was entrusted to 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 phagemid. At this time, GLS3000 was selected as the L chain. The constructed phage display phagemid was then introduced into Escherichia coli by electroporation to produce Escherichia coli with antibody library fragments.

[0562] [Table 13]

[0563]

[0564] [Table 14]

[0565]

[0566] [Example 7] Obtaining Fab domains binding to CD3 and the second antigen (IL6R) from the Dual Fab library

[0567] (7-1) Obtaining the Fab domain that binds to human IL6R

[0568] Fab domains (antibody fragments) binding to human IL6R were identified based on the Dual Fab library designed and constructed in Example 6. Using biotin-labeled human IL6R as an antigen, antibody fragments capable of binding to human IL6R were enriched.

[0569] Phages are produced by the constructed Escherichia coli that retains phage display phagemids. The phage population precipitated by adding 2.5M NaCl / 10% PEG to the culture solution of the Escherichia coli that produced the phages was diluted with TBS to obtain a phage library solution. Next, BSA was added to the phage library solution to a final concentration of 4% BSA. As a screening method, a screening method using antigens immobilized on magnetic beads as a conventional method 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). As magnetic beads, NeutrAvidin-coated magnetic beads (NeutrAvidin-coated Sera-Mag SpeedBeads) or streptavidin-coated magnetic beads (Dynabeads M-280 Streptavidin) were used.

[0570] Specifically, by adding 250 pmol of biotin-labeled antigen to the modulated phage library solution, 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 magnetic beads were washed 3 times with TBST (TBS containing 0.1% Tween20, TBS manufactured by TaKaRa) and then washed 2 times with 1 mL of TBS. After that, the magnetic beads added with 0.5 mL of 1 mg / mL trypsin were suspended at room temperature for 15 minutes, and then the magnetic beads were immediately separated using a magnetic stand to recover the phage solution. The recovered phage solution was added to 10 mL of Escherichia coli strain ER2738 in the logarithmic growth phase (OD600 of 0.4-0.5). The above-mentioned Escherichia coli was slowly stirred and cultured at 37°C for 1 hour to infect the Escherichia coli with phage. The infected Escherichia coli was inoculated on a 225mm×225mm plate. Next, a phage library solution was prepared by recovering phages from the culture solution of the inoculated Escherichia coli. This cycle is called screening and is repeated many times. It should be noted that 40pmol of biotin-labeled antigen was used in the screening after the second time. In addition, in the fourth screening, phages were concentrated using binding to CD3 as an indicator. Specifically, 250pmol of biotin-labeled CD3ε peptide antigen (amino acid sequence SEQ ID NO:60) was added to the modulated phage library solution, and the phage library was contacted with the antigen at room temperature for 60 minutes. Magnetic beads blocked with BSA were added to allow the antigen-phage complex to bind to the magnetic beads at room temperature for 15 minutes. The magnetic beads were washed with 1mL of TBS and TBS containing 0.1% Tween20. The magnetic beads to which 0.5 mL of 1 mg / mL trypsin was added were suspended at room temperature for 15 minutes, and then the magnetic beads were immediately separated using a magnetic stand to recover the phage solution. The phage recovered from the phage solution treated with trypsin was added to 10 mL of Escherichia coli strain ER2738 in the logarithmic growth phase (OD600 of 0.4-0.7). The above-mentioned Escherichia coli was slowly stirred and cultured at 37°C for 1 hour to infect the Escherichia coli with the phage. The infected Escherichia coli was inoculated on a 225 mm × 225 mm plate. Next, the phage was recovered from the culture solution of the inoculated Escherichia coli, thereby recovering the phage library solution.

[0571] Furthermore, in order to prevent multiple phages from infecting one E. coli, the phage library solution prepared from E. coli infected with phages recovered in the fifth screening was diluted 100,000 times again, and the resulting phage solution was used to infect E. coli to obtain a single colony.

[0572] (7-2) Binding of Fab domain presented by phage to CD3 or IL6R (phage ELISA method) According to the conventional method (Methods Mol.Biol.(2002)178,133-145), the culture supernatant containing phage is recovered from the single colony of Escherichia coli obtained by the above method. The culture supernatant containing phage to which BSA is added to make the final concentration of BSA 4% is provided to ELISA according to the following order. A StreptaWell 96 microtiter plate (Roche) is 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. The antigen is removed by washing each well of the plate with PBST, and then the well is blocked with 250 μL of 4% BSA-TBS for more than 1 hour. The plate with the prepared culture supernatant added to each well from which 4% BSA-TBS was removed was left to stand at room temperature for 1 hour to allow the antibody presenting the phage to bind to the antigen present in each well. Each well was washed with TBST, and then HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted in TBS with a final concentration of 4% BSA was added and incubated for 1 hour. After washing with TBST, the color development reaction of the solution in each well to which TMB single solution (ZYMED) was added was stopped by adding sulfuric acid, and then the color development was measured based on the absorbance at 450nm. The results are shown in Fig.19 . The results showed that clones #50 and #62 have binding properties to CD3ε and human IL6R. That is, by using the Dual Fab library, clones that show binding properties to the second antigen (human IL6R in Example 7) can be selected. By further increasing the number of evaluations, selecting clones that show binding properties and converting them into IgG (connecting the VH and VL sequences possessed by the clones to the human H chain or L chain constant region, respectively), their binding properties to CD3ε and the second antigen (human IL6R) can be evaluated. Furthermore, by the method described in Example 3 or 4 or the competition method, it is possible to study whether they bind to CD3ε and the second antigen (human IL6R) at the same time. The competition method shows that, for example, in the presence of the second antigen, the binding of the clone to CD3ε is reduced compared to when the antibody is present alone, so it does not bind simultaneously.

[0573] [Example 8] Obtaining a Fab domain that binds to CD3 and a second antigen (human IgA) from a dual Fab library (8-1) Obtaining a Fab domain that binds to human IgA

[0574] IgA is an antibody isotype that exists in large quantities in the body and is known as a molecule involved in biological defense at the intestinal tract or mucosal surfaces. It is known to bind to FcαR (Fcα receptor) (J. Pathol. 208: 270-282, 2006).

[0575] Fab domains (antibody fragments) binding 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 an antigen, antibody fragments capable of binding to human IgA were enriched.

[0576] Phages were produced by the constructed Escherichia coli that retained the phagemid for phage display. The phage group precipitated by adding 2.5M NaCl / 10% PEG to the culture solution of the Escherichia coli that produced the phage was diluted with TBS to obtain a phage library solution. Next, BSA was added to the phage library solution to a final concentration of 4% BSA. As a screening method, reference was made to the screening method using antigens immobilized on magnetic beads as a conventional method (J. Immunol. Methods. (2008) 332 (1-2), 2-9; J. Immunol. Methods. (2001) 247 (1-2), 191-203; Biotechnol. Prog. (2002) 18 (2) 212-20; Mol. Cell Proteomics (2003) 2 (2), 61-9). As magnetic beads, NeutrAvidin-coated magnetic beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or streptavidin-coated magnetic beads (Dynabeads M-280 Streptavidin) were used.

[0577] Specifically, by adding 250 pmol of biotin-labeled antigen to the modulated phage library solution, 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 magnetic beads were washed 3 times with TBST (TBS containing 0.1% Tween20, TBS manufactured by TaKaRa) and then washed 2 times with 1 mL of TBS. After that, the magnetic beads added with 0.5 mL of 1 mg / mL trypsin were suspended at room temperature for 15 minutes, and then the magnetic beads were immediately separated using a magnetic stand to recover the phage solution. The recovered phage solution was added to 10 mL of Escherichia coli strain ER2738 in the logarithmic growth phase (OD600 of 0.4-0.5). The above-mentioned Escherichia coli was slowly stirred and cultured at 37°C for 1 hour to infect the Escherichia coli with phage. The infected E. coli was then plated on a 225 mm x 225 mm plate. Next, the phage library solution was prepared by recovering the phage from the culture solution of the inoculated E. coli. This cycle was called screening and was repeated 4 times. It should be noted that human IgA was set to 40 pmol in the second and subsequent screenings.

[0578] (8-2) Binding of Fab domain presented by phage to CD3 or human IgA

[0579] According to conventional methods (Methods Mol.Biol.(2002)178,133-145), the culture supernatant containing phages was recovered from the single colony of Escherichia coli obtained by the above method. The culture supernatant containing phages to which BSA was added to make the final concentration of 4% BSA was provided to ELISA in the following order. StreptaWell 96 microtiter plates (Roche) were coated with 100 μL of PBS containing biotinylated antigen (biotinylated CD3ε peptide or biotinylated human IgA, refer to Example 3) at 4°C overnight, or at room temperature for 1 hour. The antigen was removed by washing each well of the plate with PBST, and then the wells were blocked with 250 μL of 0.1×TBS / 150mM NaCl / 0.02% skim milk for more than 1 hour. The plate to which the prepared culture supernatant was added after removing 0.1×TBS / 150mM NaCl / 0.02% skim milk was left to stand at room temperature for 1 hour to allow the antibodies presented by the phage to bind to the antigens present in each well. Each well was washed with 0.1×TBS / 150mM NaCl / 0.01% Tween20, and then HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted with 0.1×TBS / 150mM NaCl / 0.01% Tween20 was added and incubated for 1 hour. After washing with TBST, the color development reaction of the solution in each well to which TMB single solution (ZYMED) was added was stopped by adding sulfuric acid, and then the color development was measured based on the absorbance at 450nm. The results are shown in Fig. 20 .like Fig. 20 As shown, there are clones that bind to CD3 and human IgA, and by using the Dual Fab library, clones that show binding to the second antigen (human IgA in Example 8) can be selected.

[0580] (8-3) Binding of IgG Having the Acquired Fab Domain to CD3 or Human IgA

[0581] For the clones that showed binding to CD3 and human IgA in (8-2), the VH fragment was amplified from Escherichia coli having the sequence of the above clone by PCR using primers that specifically bind to the H chain of the Dual Fab library. The amplified VH fragment was inserted into the plasmid for animal cell expression into which pE22Hh was inserted according to the method of Reference Example 1, and was expressed and purified in the form of a one-armed antibody in the same manner as in Example 6 (6-2). The clone name and SEQ ID NO (sequence number) of the H chain sequence are shown in Table 15. That is, the sequence obtained by connecting the H chain shown in Table 15, Kn010G3 (SEQ ID NO: 56), GLS3000 (SEQ ID NO: 53) as the L chain, and the Kappa sequence (SEQ ID NO: 55) was used, and expression and purification were performed according to Reference Example 1.

[0582] [Table 15]

[0583] Sample name SEQ ID NO IGAR4C09#1 61 IGAR4C12#4 62 IGAR6(2)B02#1 63

[0584] The electrochemiluminescence method (ECL method) was used to determine whether the antibody molecule having the obtained Fab region binds to CD3ε and human IgA. Specifically, 25 μL of a biotinylated CD3ε peptide (described in Example 7) or a biotinylated human IgA (reference example 3) diluted with a TBST solution (manufactured by TaKaRa, a solution obtained by adding 0.1% Tween20 to TBS) was added to each well of a Nunc-Immuno(tm) MicroWell(tm) 96-well circular plate (Nunc), an antibody solution prepared to 2 μg / mL, and an anti-human IgG antibody (Invitrogen #628400) to which a sulfo-tag was added were mixed, and then incubated at room temperature for more than 1 hour while shielded from light to form an antibody-antigen complex. 150 μL of a TBST solution containing 0.5% BSA (labeled as a blocking solution) was added to each well of a streptavidin plate (MSD), and incubated at room temperature for more than 1 hour. After removing the blocking solution, wash 3 times with 250μL TBS(-) solution. Add 50μL of antibody-antigen complex solution to each well and incubate at room temperature for 1 hour to bind the biotinylated antigen-antibody-sulfo-tag antibody complex solution to the streptavidin plate via the biotinylated antigen. After removing the antibody-antigen complex solution, wash 3 times with TBST solution, add 150μL of a solution obtained by diluting 4×READ buffer (MSD) 2 times with water to each well, and detect the luminescent signal of sulfo-tag by Sector Imager2400 (MSD).

[0585] The results are shown in Fig.21The results showed that when clones that were confirmed to bind by phage ELISA (including clones containing some amino acid mutations) were converted to IgG, even if the sequences that were confirmed to bind by phage ELISA were converted to IgG, these sequences also bound to CD3ε and human IgA.

[0586] This result shows that antibodies that bind to the second antigen can be obtained from the Dual Fab library, and clones that can be concentrated (enriched) even if there is only a Fab domain in the panning using the general phage library, but binding is confirmed even when an IgG containing an Fc region is formed. Therefore, the Dual Fab library is considered to be a library that can obtain a Fab domain that retains the ability to bind to CD3 and has the ability to bind to the second antigen.

[0587] (8-4) Evaluation of simultaneous binding of IgG with the acquired Fab domain to CD3 (CD3ε) and human IgA (8-3) showed that the clones obtained from the Dual Fab library had binding activity even when formed into IgG. Next, the competition method (electrochemiluminescence method (ECL method)) was used to determine whether the obtained IgG simultaneously bound to CD3 (CD3ε) and human IgA. When binding to CD3 (CD3ε) and human IgA simultaneously, the ECL signal did not change even if CD3 (CD3ε) was added to the antibody that bound to IgA, but when simultaneous binding was not possible, if CD3 (CD3ε) was added, a part of the antibody would bind to CD3 (CD3ε) and the ECL signal should decrease.

[0588] Specifically, 25 μL of biotinylated human IgA diluted with 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 anti-human IgG antibody (Invitrogen #628400) with sulfo-tag added were added to each well of Nunc-Immuno(tm) MicroWell(tm) 96-well circular plate (Nunc), mixed, and then incubated at room temperature for more than 1 hour while shielded from light to form an antibody-antigen complex. 150 μL of TBST solution containing 0.5% BSA (labeled as blocking solution. TBST solution is a solution obtained by adding 0.1% Tween20 to TBS manufactured by TaKaRa) was added to each well of the streptavidin plate (MSD), and incubated at room temperature for more than 1 hour. After removing the blocking solution, wash 3 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-sulfo-tag antibody complex solution to bind to the streptavidin plate via the biotinylated antigen. After removing the antibody-antigen complex solution, the plate was washed three times with TBST solution, and 150 μL of the solution obtained by diluting the 4×READ buffer (MSD) 2 times with water was added to each well, and the luminescent signal of the sulfo-tag was detected by Sector Imager 2400 (MSD).

[0589] The results are shown in Fig. 22 . When the CD3ε homodimer protein was added for competition, a decrease in the ECL signal was confirmed compared to when TBST was added. The results show that the molecules that bind to CD3 and human IgA discovered through this study are Dual Fab molecules that cannot bind to human IgA if they bind to CD3. The results show that antibodies with the ability to bind to a second antigen can be obtained from the Dual Fab library, among which Dual Fab molecules that cannot bind to multiple antigens at the same time that cannot bind to the second antigen if they bind to CD3 (or cannot bind to CD3 if they bind to the second antigen) can be obtained.

[0590] When a binding molecule is found by binding activity evaluation using phage in (8-2), it is obvious to those skilled in the art that the diversity of the sequence of the binding molecule can be increased by increasing the number of evaluations, and therefore, it can be seen from the above results that the Dual Fab library is a library that can obtain Fab domains that have the ability to bind to the second antigen while maintaining the ability to bind to CD3. In addition, although a Dual Fab library in which only the H chain is diversified is used in this example, generally, a library with a larger size (also called diversity, which means that a library contains a variety of sequences) is more likely to obtain antigen-binding molecules, and therefore, as in the case shown in this example, a Dual Fab library in which the L chain is also diversified can also be used to obtain Dual Fab molecules.

[0591] As shown in Example 8, if Dual Fab molecules can be prepared, Fab or antigen-binding domains that bind to the third antigen can be identified by methods known to those skilled in the art, such as the hybridoma method or a method for screening binding antibodies (or binding domains) from an antibody library. For antibodies having the identified antigen-binding domain (e.g., Fab) that binds to the third antigen and the Fab domain of the Dual Fab molecule, multispecific antibodies can be obtained by methods known to those skilled in the art for preparing multispecific antibodies, such as a method for preparing two antibodies with different H chains by making the L chain common (a technique for controlling the interface of each domain of the Fc region), the Cross Mab method, and the Fab arm exchange method. That is, if Dual Fab molecules can be identified, the desired multispecific antibody can be obtained by combining the Fab that binds to the third antigen and the Dual Fab that binds to the first antigen and the second antigen shown in Example 8 by methods known to those skilled in the art.

[0592] (8-5) About CD3 / human IgA Dual Fab molecule

[0593] Example 8 shows that a Dual Fab molecule that binds to CD3ε and human IgA but does not bind to both CD3ε and human IgA simultaneously can be obtained. Furthermore, an antigen-binding domain that binds to a third antigen can be added by methods known to those skilled in the art.

[0594] In recent years, studies have 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). It has been reported that the principle is that FcαR, which is an IgA receptor, is expressed in polymorphonuclear cells and induces autophagy in cancer cells (J Immunol 2011; 187: 726-732). This example clearly shows that Dual Fab molecules that bind to CD3 and IgA can be constructed, but when molecules that bind to FcαR via IgA are explored by methods known to those skilled in the art (e.g., ELISA or ECL method), anti-tumor effects mediated by FcαR can be expected. That is, the Dual Fab can induce cytotoxic activity produced by T cells by binding to CD3ε in cells expressing any third antigen, and induce cytotoxic activity produced by cells expressing FcαR via binding to IgA, and strong cytotoxic activity can be expected.

[0595] [Example 9] Obtaining Fab domains binding to CD3 and a second antigen (human CD154) from a dual Fab library

[0596] (9-1) Obtaining the Fab domain that binds to human CD154

[0597] Fab domains (antibody fragments) binding to human CD154 were identified based on the Dual Fab library designed and constructed in Example 6. Using biotin-labeled human CD154 as an antigen, antibody fragments capable of binding to human CD154 were enriched.

[0598] Phages were produced by the constructed Escherichia coli that retained the phagemid for phage display. The phage group precipitated by adding 2.5M NaCl / 10% PEG to the culture solution of the Escherichia coli that produced the phage was diluted with TBS to obtain a phage library solution. Next, BSA was added to the phage library solution to a final concentration of 4% BSA. As a screening method, reference was made to a screening method using antigens immobilized on magnetic beads as a conventional method (J. Immunol. Methods. (2008) 332 (1-2), 2-9; J. Immunol. Methods. (2001) 247 (1-2), 191-203; Biotechnol. Prog. (2002) 18 (2) 212-20; Mol. Cell Proteomics (2003) 2 (2), 61-9). As magnetic beads, NeutrAvidin-coated magnetic beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or streptavidin-coated magnetic beads (Dynabeads M-280 Streptavidin) were used.

[0599] Specifically, by adding 250 pmol of biotin-labeled antigen to the modulated phage library solution, 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 magnetic beads were washed 3 times with TBST (TBS containing 0.1% Tween20, TBS manufactured by TaKaRa) and then washed 2 times with 1 mL of TBS. After that, the magnetic beads added with 0.5 mL of 1 mg / mL trypsin were suspended at room temperature for 15 minutes, and the magnetic beads were immediately separated using a magnetic stand to recover the phage solution. The recovered phage solution was added to 10 mL of Escherichia coli strain ER2738 in the logarithmic growth phase (OD600 of 0.4-0.5). The above-mentioned Escherichia coli was slowly stirred and cultured at 37°C for 1 hour, so that the phage infected the Escherichia coli. The infected E. coli was inoculated on a 225 mm × 225 mm plate. Next, the phage library solution was prepared by recovering the phage from the culture solution of the inoculated E. coli. This cycle is called screening and was repeated 5 times. It should be noted that human CD154 was set to 40 pmol in the second and subsequent screenings.

[0600] (9-2) Binding of Fab domain presented by phage to CD3 or human CD154

[0601] According to conventional methods (Methods Mol.Biol.(2002)178,133-145), the culture supernatant containing phages was recovered from a single colony of Escherichia coli obtained by the above method. The culture supernatant containing phages to which BSA was added to make the final concentration of 4% BSA was provided to ELISA in the following order. 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. The antigens were removed by washing each well of the plate with PBST, and then the wells were blocked with 250 μL of 0.1×TBS / 150mM NaCl / 0.02% skim milk for more than 1 hour. After removing 0.1×TBS / 150mM NaCl / 0.02% skim milk, the plate to which the prepared culture supernatant was added was left to stand at room temperature for 1 hour to allow the antibody presenting the phage to bind to the antigen present in each well. Each well was washed with 0.1×TBS / 150mM NaCl / 0.01% Tween20, and then HRP-conjugated anti-M13 antibody (AmershamPharmacia Biotech) diluted with 0.1×TBS / 150mM NaCl / 0.01% Tween20 was added and incubated for 1 hour. After washing with TBST, the color development reaction of the solution in each well to which TMB single solution (ZYMED) was added was stopped by adding sulfuric acid, and then the color development was measured based on the absorbance at 450nm. The results are shown in Fig.23 .like Fig.23 As shown in the examples, it was shown that there were clones that bind to CD3 and CD154, and that clones that bind to the second antigen (human CD154 in Example 9) could be selected by using the Dual Fab library. In addition, as shown in Examples 7, 8, and 9, binding Fab domains could be obtained for three different antigens, thereby showing that the Dual Fab library functions as a library for obtaining molecules that bind to the second antigen.

[0602] (9-3) Binding of IgG Having the Acquired Fab Domain to CD3 or Human CD154

[0603] For the clones shown to bind to CD3 and human CD154 in (9-2), the VH fragment was amplified by PCR from Escherichia coli having the sequence of the above clone using primers that specifically bind to the H chain of the Dual Fab library. The amplified VH fragment was inserted into the plasmid for animal cell expression into which pE22Hh was inserted according to the method of Reference Example 1, and expressed and purified in the form of a one-armed antibody in the same manner as in Example 6 (6-2). The obtained sequence name and SEQ ID NO (sequence number) of the H chain sequence are described in Table 16. Specifically, the sequence in which the H chain and Kn010G3 (SEQ ID NO: 56) described in Table 16 were connected to GLS3000 (SEQ ID NO: 53) and Kappa sequence (SEQ ID NO: 55) as the L chain was expressed and purified according to Reference Example 1.

[0604] [Table 16]

[0605]

[0606] The electrochemiluminescence method (ECL method) was used to determine whether the antibody molecule having the Fab region shown to bind to CD3 and human CD154 by the phage ELISA method in (9-2) binds to CD3 and human CD154. Specifically, 25 μL of biotinylated CD3 or biotinylated human CD154 diluted with TBST solution, 25 μL of antibody solution prepared to 2 μg / mL, and 25 μL of anti-human IgG antibody (Invitrogen #628400) added with sulfo-tag were added to each well of a Nunc-Immuno (tm) MicroWell (tm) 96-well circular plate (Nunc) and mixed, and then incubated at room temperature for more than 1 hour while shielded from light to form an antibody-antigen complex. Add 150 μL of TBST solution containing 0.5% BSA (marked as blocking solution. TBST solution is a solution obtained by adding 0.1% Tween20 to TBS manufactured by TaKaRa) to each well of the streptavidin plate (MSD), and incubate at room temperature for more than 1 hour. After removing the blocking solution, wash 3 times with 250 μL of TBST solution. Add 50 μL of antibody-antigen complex solution to each well, incubate at room temperature for 1 hour, and allow the complex solution of biotinylated antigen-antibody-sulfo-tag antibody for detection to bind to the streptavidin plate via the biotinylated antigen. After removing the antibody-antigen complex solution, wash 3 times with TBST solution, add 150 μL of a solution obtained by diluting 4×READ buffer (MSD) 2 times with water to each well, and detect the luminescent signal of sulfo-tag by Sector Imager 2400 (MSD).

[0607] The results are shown in Fig.24 The results showed that when clones that were confirmed to bind by phage ELISA (including clones containing some amino acid mutations) were converted to IgG, even if the sequences that were confirmed to bind by phage ELISA were converted to IgG, these sequences also bound to CD3ε and human CD154.

[0608] This result shows that antibodies binding to the second antigen can be obtained from the Dual Fab library, and in the panning using the general phage library, although only the Fab domain was present, it was confirmed that the enrichment of clones that bind to not only human IgA but also human CD154 was formed even when IgG containing the Fc region was formed. Therefore, the Dual Fab library is considered to be a library that can obtain Fab domains that have the ability to bind to the second antigen while maintaining the ability to bind to CD3.

[0609] (9-4) Evaluation of simultaneous binding of IgG with the acquired Fab domain to CD3ε and human CD154 (9-3) showed that the clones obtained from the Dual Fab library bind even when they are formed into IgG. Next, the competition method (electrochemiluminescence method (ECL method)) was used to determine whether the obtained IgG binds to CD3 and human CD154 at the same time. When binding to CD3 and human CD154 at the same time, the ECL signal does not change even if CD3 is added to the antibody that binds to CD154, but when simultaneous binding is not possible, if CD3 is added, a part of the antibody binds to CD3 and the ECL signal should decrease.

[0610] Specifically, 25 μL of biotinylated human CD154 diluted with 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 anti-human IgG antibody (Invitrogen #628400) added with sulfo-tag were added to each well of Nunc-Immuno (tm) MicroWell (tm) 96-well circular plate (Nunc), mixed, and then incubated at room temperature for more than 1 hour while shielded from light to form an antibody-antigen complex. 150 μL of TBST solution containing 0.5% BSA (labeled as blocking solution. TBST solution is a solution obtained by adding 0.1% Tween20 to TBS manufactured by TaKaRa) was added to each well of the streptavidin plate (MSD), and incubated at room temperature for more than 1 hour. After removing the blocking solution, wash 3 times with 250μL TBST solution. Add 50μL of antibody-antigen complex solution to each well, incubate at room temperature for 1 hour, and bind the biotinylated antigen-antibody-sulfo-tag antibody complex solution to the streptavidin plate via the biotinylated antigen. After removing the antibody-antigen complex solution, wash 3 times with TBST solution, add 150μL of a solution obtained by diluting 4×READ buffer (MSD) 2 times with water to each well, and detect the luminescent signal of sulfo-tag by Sector Imager 2400 (MSD).

[0611] The results are shown in Fig.25 . When the CD3ε homodimer protein was added for competition, a decrease in the ECL signal was confirmed compared to when TBST was added. The results show that the molecules that bind to CD3ε and human CD154 discovered in this study are Dual Fab molecules that cannot bind to human CD154 if they bind to CD3. The results show that antibodies that bind to the second antigen can be obtained from the Dual Fab library, among which Dual Fab molecules that cannot bind to multiple antigens at the same time and cannot bind to the second antigen if they bind to CD3 (or cannot bind to CD3 if they bind to the second antigen) can be obtained.

[0612] In (9-2), when a binding molecule is found by evaluating the binding activity using phage, it is obvious to those skilled in the art that the diversity of the sequence of the binding molecule can be increased by increasing the number of evaluations, and therefore, from the above results, it can be seen that the Dual Fab library is a library that can obtain Fab domains that have the ability to bind to the second antigen while maintaining the ability to bind to CD3. In addition, in this example, although a Dual Fab library in which only the H chain is diversified is used, generally, a library with a larger size (also called diversity, which means that a variety of sequences are included in the library) can obtain more antigen-binding molecules, and therefore a Dual Fab library in which the L chain is also diversified can also be used to obtain Dual Fab molecules as shown in this example.

[0613] As shown in Example 9, if Dual Fab molecules can be prepared, Fab or antigen-binding domains that bind to the third antigen can be identified by methods known to those skilled in the art, such as the hybridoma method or a method for screening binding antibodies or antigen-binding domains from an antibody library. For antibodies having the identified antigen-binding domain (e.g., Fab) that binds to the third antigen and the Fab domain of the Dual Fab molecule, multispecific antibodies can be obtained by methods known to those skilled in the art for preparing multispecific antibodies, such as a method for preparing two antibodies with different H chains by making the L chain common (a technique for controlling the interface of each domain of the Fc region), the Cross Mab method, and the Fab arm exchange method. That is, if Dual Fab molecules can be identified, the desired multispecific antibody can be obtained by combining a Fab that binds to the third antigen with the Dual Fab that binds to the first antigen and the second antigen as shown in Example 9 by methods known to those skilled in the art. The above examples show that by applying the Dual Fab library to a variety of antigens, molecules that bind to CD3ε and the second antigen can be obtained, and molecules that bind to the first antigen (CD3ε) and the second antigen but not to both the first antigen and the second antigen can be obtained by Examples 8 and 9. As described above, the identification of Fabs that bind to the third antigen can be performed by methods known to those skilled in the art, so the desired antibodies described in Example 1 can be obtained by using the Dual Fab library.

[0614] (9-5) About CD3 / human CD154 Dual Fab molecule

[0615] Example 9 shows that a Dual Fab molecule that binds to CD3ε and human CD154 but does not bind to both CD3ε and human CD154 simultaneously can be obtained. In addition, 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.

[0616] In recent years, studies have shown that agonistic antibodies to CD40, which is a CD154 receptor, enhance antitumor activity in a method of transferring cancer antigen-reactive T cells (J Immunother. 2012Apr; 35(3): 276-82.). It is clear that this embodiment can construct a Dual Fab molecule that binds to CD3 and CD154, and when an antibody that exhibits agonistic activity to CD40 via CD154 is selected, an antitumor effect mediated by CD40 can be expected. That is, it can be expected that the Dual Fab can enhance the cytotoxic activity produced by T cells by binding to CD3ε in cells expressing any third antigen, and enhance the antitumor effect mediated by the agonistic signal of CD40 by binding to CD154.

[0617] [Reference Example]

[0618] [Reference Example 1] Preparation of antibody expression vector and expression and purification of antibody

[0619] The QuikChange site-directed mutagenesis kit (Stratagene), PCR or In fusion Advantage PCR cloning kit (TAKARA) etc. were used to introduce amino acid substitutions according to methods known to those skilled in the art, and an expression vector was constructed. The nucleotide sequence of the resulting expression vector was determined by methods known to those skilled in the art. The prepared plasmid was transiently introduced into HEK293H cell lines (Invitrogen) or FreeStyle293 cells (Invitrogen) derived from human embryonic kidney cancer cells to express the antibody. rProtein A Sepharose was used TM Fast Flow (GE Healthcare), according to the method known to those skilled in the art, the antibodies were purified from the obtained culture supernatant. The concentration of purified antibodies was determined by measuring the absorbance at 280 nm using a spectrophotometer, and the absorbance coefficient was calculated from the obtained value by the PACE method, and the antibody concentration was calculated using the absorbance coefficient (Protein Science 1995; 4: 2411-2423).

[0620] [Reference Example 2] Evaluation of antibody binding to ECM (Extracellular matrix)

[0621] With reference to WO2012093704A1, the binding evaluation of antibodies to ECM (Extracellular matrix) was implemented in the following order. ECM (BD Matrigel #356237) without phenol red was diluted to 2 mg / mL with TBS, and 5 μL was dropped into the center of each well of the ECL assay plate (L15XB-3, MSD high bind) cooled on ice. After that, it was covered with a sealing strip and left to stand at 4°C overnight. The plate with ECM fixed was returned to room temperature, and 150 μL of ECL blocking buffer (a solution obtained by adding 0.5% BSA and 0.05% Tween20 to PBS) was added to each well, and left to stand at room temperature for more than 2 hours or at 4°C overnight. Next, the antibody sample was diluted to 9 μg / mL using PBS-T (a solution obtained by adding 0.05% Tween20 to PBS). Use ECLDB (a solution obtained by adding 0.1% BSA and 0.01% Tween20 to PBS) to dilute the secondary antibody to 2μg / mL, add 20μL of antibody solution and 30μL of secondary antibody solution to a round-bottom plate with 10μL of ECLDB injected into each well, and stir at room temperature for 1 hour in the dark. Pour out the ECL blocking buffer from the ECM plate to which the ECL blocking buffer was added, and add 50μL of the mixed solution of the above-mentioned antibody and secondary antibody to the plate. Then let it stand at room temperature for 1 hour in the dark. After pouring out the sample, add 150μL of READ buffer (MSD) to each well, and detect the luminescent signal of sulfo-tag by Sector Imager 2400 (MSD).

[0622] [Reference Example 3] Preparation of human IgA

[0623] As human IgA, the Fc part of the naturally occurring human IgA sequence (human IgA-Fc) was used. In order 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 was added was connected via a linker by biotin ligase. The gene fragment encoding the protein (SEQ ID NO: 80) connected to the human IgA-Fc and AviTag sequences was inserted into an animal cell expression vector, and the constructed plasmid vector was introduced into FreeStyle293 cells (Invitrogen) using 293Fectin (Invitrogen). At this time, the gene expressing EBNA1 (SEQ ID NO: 81) and the gene expressing biotin ligase (BirA, SEQ ID NO: 82) were introduced at the same time, and biotin was added to biotin-label human IgA-Fc. The cells into which the genes were introduced in the above order were cultured at 37°C and 8% CO2 for 6 days to secrete the target protein into the culture supernatant.

[0624] The cell culture solution containing the target human IgA-Fc was filtered with a 0.22μm bottle-top filter to obtain the culture supernatant. The culture supernatant diluted with the same solution was loaded onto a HiTrap QHP (GE Healthcare) equilibrated with 20mM Tris-HCl, pH7.4, and the target human IgA-Fc was eluted by a NaCl concentration gradient. Next, the above HiTrap Q HP eluate diluted with the same solution was loaded onto a SoftLink Avidin column (Promega) equilibrated with 50mM Tris-HCl, pH8.0, and the target human IgA-Fc was eluted with 5mM biotin, 150mM NaCl, 50mM Tris-HCl, pH8.0. Thereafter, the aggregates as unintended impurities were removed by gel filtration chromatography using Superdex 200 (GE Healthcare), and the buffer was replaced with 20 mM histidine-HCl, 150 mM NaCl, pH 6.0 to obtain purified human IgA-Fc.

[0625] Industrial Applicability

[0626] According to the present invention, a polypeptide is provided that can enhance the activity produced by an antigen-binding molecule while avoiding cross-linking between different cells caused by binding to antigens expressed on different cells, which is considered to be the cause of side effects, and is suitable for use as a pharmaceutical.

Claims

1. A method for preparing a library of antigen-binding molecules, wherein the antigen-binding molecules are capable of binding to a first antigen and a second antigen different from the first antigen, wherein the first antigen is CD3, and the second antigen is a molecule expressed on the surface of a natural immune cell and is not CD3, wherein the antigen-binding molecules comprise an antibody variable region, wherein the antibody variable region comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH), and the antibody variable region does not bind to the first antigen and the second antigen at the same time, The method comprises the following steps: Inserting a peptide that binds to a second antigen into an antigen-binding molecule that binds to a first antigen by changing at least one amino acid in the sequence of SEQ ID NO: 13, 52, or 59, which is a template sequence of an antibody heavy chain variable domain, and wherein the change comprises a substitution or insertion of an amino acid sequence in the template sequence, and The amino acid to be changed is at least one amino acid selected from positions 52a to 52c, 72 to 73 and 99 to 100a by Kabat numbering in the heavy chain variable domain of the antibody variable region.

2. The method of claim 1, wherein: The antibody variable region that does not bind to the first antigen and the second antigen at the same time is a variable region that does not bind to the first antigen and the second antigen that are expressed on different cells.

3. The method of claim 1, wherein: The antigen binding molecule also comprises an antibody Fc region.

4. The method of claim 3, wherein: The Fc region has reduced FcγR-binding activity compared to the Fc region of a native human IgG1 antibody.

5. The method of claim 1, wherein: The antigen binding molecule is a multispecific antibody.

6. The method of claim 1, wherein: The number of inserted amino acids ranges from 1 to 25.

7. The method of claim 1, wherein: The second antigen is FcγR, TLR, IgA, lectin, immune restriction point molecule, TNF superfamily molecule, TNFR superfamily molecule or NK receptor molecule.

8. The method of claim 1, wherein the antigen-binding molecule further comprises a variable region that binds to a third antigen, and wherein the third antigen is a molecule specifically expressed in cancer tissue.

9. A library comprising a plurality of antigen-binding molecules having different amino acid sequences from each other, wherein the antigen-binding molecules comprise antibody variable regions that can bind to a first antigen and a second antigen but not simultaneously; The first antigen is CD3, and the second antigen is a molecule expressed on the surface of natural immune cells and is not CD3; Each antibody variable region comprises VH and VL, VH having a sequence having at least one amino acid change in the sequence of SEQ ID NO: 13, SEQ ID NO: 52 or SEQ ID NO: 59 as a template sequence; and in, The sequences differ at one or more amino acid positions which: (a) vary from one antibody variable region to another, and (b) selected from individual Kabat numbered VH positions 52a to 52c, 72 to 73, and 99 to 100a, and wherein the change comprises a substitution or insertion of an amino acid sequence in a template sequence that binds to the first antigen, and The change is to replace a part of the amino acid sequence of the variable region that binds to the first antigen with the amino acid sequence that binds to the second antigen, or to insert the amino acid sequence that binds to the second antigen into the amino acid sequence of the variable region that binds to the first antigen.

10. The library of claim 9, wherein the second antigen is selected from the group consisting of: FcγR, toll-like receptor (TLR), lectin, immune restriction point molecule, tumor necrosis family receptor (TNFR) superfamily molecule, and natural killer (NK) cell receptor molecule.

11. The library of claim 9, wherein The number of amino acids to be inserted is 1 to 25.

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