Dual-specific anti-CCL2 antibody

Abstract

To provide bispecific anti-CCL2 antibodies binding to two different epitopes on human CCL2, pharmaceutical compositions thereof, their manufacture, and use as medicaments for the treatment of cancers, inflammatory, autoimmune and ophthalmologic diseases.SOLUTION: A bispecific antibody comprises a first antigen-binding site that binds to a first epitope on human CCL2 and a second different antigen-binding site that binds a second different epitope on human CCL2, wherein the bispecific antibody comprises a Fc domain of human IgG isotype.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to bispecific anti-CCL2 antibodies that bind to two different epitopes on human CCL2, pharmaceutical compositions thereof, the manufacture thereof, and their use as pharmaceuticals for the treatment of cancer, inflammatory diseases, autoimmune diseases, and ophthalmic diseases. [Background technology]

[0002] background The CCL2 / CCR2 axis is the primary mediator of the recruitment of immature myeloid cells to tumors. CCL2 is overexpressed by malignant cells and binds to the extracellular matrix (ECM), which constructs a chemoattractant gradient. Once in the tumor, myeloid suppressor cells (MDSCs) contribute to a protumorogenic environment by secreting / upregulating anti-inflammatory cytokines / receptors that inhibit the initiation of antitumor T cell responses. In this way, MDSCs may reduce or even impair the effectiveness of any T cell activation therapy (Meyer et al, 2014). Therefore, specific inhibition of the recruitment of these immature myeloid cells enhances the effectiveness of checkpoint inhibitors, T cell bispecific antibodies (TCBs), or other cancer immunotherapies (CITs). Furthermore, CCL2 is also involved in promoting angiogenesis, metastasis, and tumor growth, suggesting that neutralizing CCL2 may contribute to several antitumor interventions.

[0003] Targeting CCL2, in contrast to its receptor, specifically inhibits undesirable CCL2-mediated effects and saves signaling through the same receptor (CCL2) but different ligands (e.g., CCL7, CCL8, CCL13) that are involved in the recruitment of other immune cell populations such as Th1 and NK cells.

[0004] Clinically, CCL2 has been a preferred antibody target in several studies aimed at neutralizing its elevated levels caused by various inflammatory diseases such as rheumatoid arthritis (Haringman et al, Arthritis Rheum. 2006 Aug;54(8):2387-92), idiopathic pulmonary fibrosis (Raghu et al, Eur Respir J. 2015 Dec;46(6):1740-50), diabetic nephropathy (Menne et al, Nephrol Dial Transplant (2017) 32:307-315), and cancer (Sandhu et al, Cancer Chemother Pharmacol. 2013 Apr;71(4):1041-50). However, its high synthesis rate, along with the observed high in vivo antibody-antigen dissociation constant (KD), has proven to be a major obstacle preventing the suppression of free CCL2 by conventional antibodies at clinically viable doses (Fetterly et al, J Clin Pharmacol. 2013 Oct;53(10):1020-7).

[0005] CCL2 neutralization has been observed in several types of cancer, including breast cancer (BC), ovarian cancer (OvCa), colorectal cancer (CRC), pancreatic cancer, and prostate cancer, and appears to be more clearly associated with elevated serum levels of CCL2 in patients. However, even in patients within these indications, where the tumor is highly infiltrated by myeloid immune cells, without presenting this serological evidence, patients can still greatly benefit from this novel therapy due to the many roles that CCL2 plays in the tumor situation, as described above.

[0006] Igawa et al, Immunological Reviews 270 (2016) 132-151, describe a sweeping technique in which the generated antibody has a pH-dependent CDR (leading to antigen degradation due to antibody-antigen dissociation in acidic endosomes), an optimized isoelectric point (pI) and enhanced binding to Fc gamma RIIb (promoting cellular uptake of immune complexes), and an engineered Fc moiety with moderate affinity for the neonatal Fc receptor, while maintaining an acceptable pharmacokinetic profile. [Overview of the project]

[0007] Summary of the Invention The present invention relates to bispecific anti-CCL2 antibodies that bind to two different epitopes on human CCL2, pharmaceutical compositions thereof, the manufacture thereof, and their use as pharmaceuticals for the treatment of cancer, inflammatory diseases, autoimmune diseases, and ophthalmic diseases.

[0008] One embodiment of the present invention is a bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a different second antigen-binding site that (specifically) binds to a different second epitope on human CCL2, and is a bispecific antibody comprising the Fc domain of a human IgG isotype.

[0009] One embodiment of the present invention is a bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a different second antigen-binding site that (specifically) binds to a second epitope on human CCL2, A)i) The first antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 33, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 34, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 35, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 36, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 38, comprising a VL domain. Includes, ii) The second antigen-binding site described above is (a) a VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 43; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46, comprising the VL domain. including, or B)i) The first antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 33, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 34, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 35, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 36, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 38, comprising a VL domain. Includes, ii) The second antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 17, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 18, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 19, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 20, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 21, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 22, comprising a VL domain. including, or C)i) The first antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 33, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 34, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 35, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 36, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 38, comprising a VL domain. Includes, ii) The second antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 9, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 11, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 12, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 13, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 14, comprising the VL domain. including, or D)i) The first antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 17, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 18, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 19, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 20, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 21, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 22, comprising a VL domain. Includes, ii) The second antigen-binding site described above is (a) a VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 43; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46, comprising the VL domain. including, or E)i) The first antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 27, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 28, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 30, comprising a VL domain. Includes, ii) The second antigen-binding site described above is (a) a VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 43; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46, comprising the VL domain. including, or F)i) The first antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 49, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 50, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 51, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 52, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 53, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 54, comprising the VL domain. Includes, ii) The second antigen-binding site described above is (a) a VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 43; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46, comprising the VL domain. including, or G)i) The first antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 9, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 11, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 12, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 13, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 14, comprising the VL domain. Includes, ii) The second antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 17, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 18, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 19, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 20, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 21, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 22, comprising a VL domain. including, or H)i) The first antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 9, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 11, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 12, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 13, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 14, comprising the VL domain. Includes, ii) The second antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 27, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 28, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 30, comprising a VL domain. including, or I)i) The first antigen-binding site described above is (a) a VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 3; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 4, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 6, comprising a VL domain. Includes, ii) The second antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 27, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 28, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 30, comprising a VL domain. It is a bispecific antibody that includes [specific component].

[0010] In one embodiment, the bispecific antibody contains the Fc domain of a human IgG isotype.

[0011] In one embodiment, the bispecific antibody comprises the Fc domain of a human IgG1 isotype.

[0012] In one embodiment, the bispecific antibody contains a constant heavy chain domain of a human IgG isotype.

[0013] In one embodiment, the bispecific antibody contains a constant heavy chain domain of a human IgG1 isotype.

[0014] In one embodiment, the in vivo clearance rate (ml / day / kg) of human CCL2 after administration of a bispecific antibody containing the constant heavy chain domain (or its Fc domain) of a human wild-type IgG1 isotype was at least twice as high (at least five times higher in one embodiment, at least ten times higher in another embodiment, and at least twenty times higher in another embodiment) when a pre-formed immune complex consisting of 20 mg / kg of each bispecific antibody and 0.1 mg / kg of human CCL2 was administered to FcRn transgenic mice at a single dose of 10 ml / kg, compared to the in vivo clearance rate (ml / day / kg) of human CCL2 after administration of a bispecific antibody containing the Fc gamma receptor silencing constant heavy chain domain (or its Fc domain) of a human IgG1 isotype including mutant L234A, L235A, and P329G (Kabat EU numbering).

[0015] One embodiment of the present invention is an isolated bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, i) The first antigen-binding site described above is A VH domain comprising the amino acid sequence of SEQ ID NO: 39, wherein (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 33, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 34, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 35; and A VL domain comprising the amino acid sequence of SEQ ID NO: 40, wherein (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 36, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 37, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 38. The antibody containing the antibody binds to the same epitope on CCL2, ii) The second antigen-binding site described above is A VH domain comprising the amino acid sequence of SEQ ID NO: 47, wherein (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 41, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 43; and A VL domain comprising the amino acid sequence of SEQ ID NO: 48, wherein (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 44, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46. This is a bispecific antibody that binds to the same epitope on CCL2 as antibodies containing [specific antibody name].

[0016] In one embodiment, the in vivo clearance rate (ml / day / kg) of human CCL2 after administration of a bispecific antibody containing the constant heavy chain domain (or its Fc domain) of a human wild-type IgG1 isotype was at least 15 times higher, and particularly at least 20 times higher, when a pre-formed immune complex consisting of 20 mg / kg of each bispecific antibody and 0.1 mg / kg of human CCL2 was administered to FcRn transgenic mice in a single dose of 10 ml / kg, compared to the in vivo clearance rate (ml / day / kg) of human CCL2 after administration of a bispecific antibody containing the constant heavy chain domain (or its Fc domain) of a human IgG1 isotype including mutant L234A, L235A, and P329G (Kabat EU numbering).

[0017] One embodiment of the present invention is an isolated bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, i) The first antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, (b) X 1 is V, I or H, and X 2 is P or H, X 3The amino acid sequence GX of SEQ ID NO: 58, where X is H or G 1 IX 2 IFX 3 A VH domain comprising a CDR-H2 comprising TANYAQKFQG, and (c) a CDR-H3 comprising the amino acid sequence YDAHYGELDF of SEQ ID NO: 59 and (d) A CDR-L1 comprising the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) a CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) a CDR-L3 comprising the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62, a VL domain comprising ii) the second antigen-binding site is (a) A CDR-H1 comprising the amino acid sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) a CDR-H3 comprising the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E, a VH domain and (d) X 1 is F or T, and X 2 is R or L, the amino acid sequence KAX 1 EDIYNRX 2 A CDR-L1 comprising A, (e) a CDR-L2 comprising the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3 comprising the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, a VL domain comprising, a bispecific antibody

[0018] One embodiment of the present invention is an (isolated) bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, i) the first antigen-binding site is (a) A CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, (b) X 1 is V, I or H, and X2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) CDR-H2 containing TANYAQKFQG, (d) CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59, (e) FR-H1 containing the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTF of SEQ ID NO: 63, (f) FR-H2 containing the amino acid sequence WVRQAPGQGLEWMG of SEQ ID NO: 64, (g) FR-H3 containing the amino acid sequence RVTITADESTSTAYMELSSLRSEDTAVY YCAR of SEQ ID NO: 65, and (g) FR-H4 containing the amino acid sequence WGQGTLVTVSS of SEQ ID NO: 66, the VH domain; and (h) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60; (i) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61; and (j) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62; (k) FR-L1 containing the amino acid sequence EIVLTQSPATLSLSPGERATLSC of SEQ ID NO: 67; (l) FR-L2 containing the amino acid sequence WYQQKPGQAPRLLIY of SEQ ID NO: 68; (m) FR-L3 containing the amino acid sequence GVPARFSGSGSGTDFTLTISSLEPEDFAVYYC of SEQ ID NO: 69; and (n) FR-L4 containing the amino acid sequence GQGTKVEIK of SEQ ID NO: 70, comprising the VL domain. Includes; ii) The second antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76; (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E; (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; (d) FR-H1 containing the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGLTIS of SEQ ID NO: 82; (e) FR-H2 containing the amino acid sequence WVRQAPGQGLEWMG of SEQ ID NO: 83; (f) FR-H3 containing the amino acid sequence RVTITADTSTSTAYMELSSLRSEDTAVYYCAR of SEQ ID NO: 84; and (g) FR-H4 containing the amino acid sequence WGQGTTVTVSS of SEQ ID NO: 85; and (h)X 1 is F or T, X 2 The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 VL domains comprising: (i) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80; (j) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R; (k) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC of SEQ ID NO: 86; (l) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIH of SEQ ID NO: 87; (m) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC of SEQ ID NO: 88; and (n) FR-L4 containing the amino acid sequence FGGGTKVEIK of SEQ ID NO: 89. It is a bispecific antibody that includes [specific component].

[0019] One embodiment of the present invention is an isolated bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, A)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 71; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 90; and VL domain containing the amino acid sequence of SEQ ID NO: 93 including, or B)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 71; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 91; and VL domain containing the amino acid sequence of SEQ ID NO: 93 including, or C)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 71; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 90; and VL domain containing the amino acid sequence of SEQ ID NO: 94 including, or D)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 72; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 90; and VL domain containing the amino acid sequence of SEQ ID NO: 94 including, or E)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 73; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 90; and VL domain containing the amino acid sequence of SEQ ID NO: 93 including, or F)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 73; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 90; and VL domain containing the amino acid sequence of SEQ ID NO: 94 including, or G)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 73; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 92; and VL domain containing the amino acid sequence of SEQ ID NO: 93 including, or H)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 73; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 91; and VL domain containing the amino acid sequence of SEQ ID NO: 93 including, or I) i) The first antigen-binding site comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 72; and a VL domain comprising the amino acid sequence of SEQ ID NO: 75 and ii) The second antigen-binding site comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 90; and a VL domain comprising the amino acid sequence of SEQ ID NO: 93 or or J) i) The first antigen-binding site comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 72; and a VL domain comprising the amino acid sequence of SEQ ID NO: 75 and ii) The second antigen-binding site comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 92; and a VL domain comprising the amino acid sequence of SEQ ID NO: 93 or or K) i) The first antigen-binding site comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 72; and a VL domain comprising the amino acid sequence of SEQ ID NO: 75 and ii) The second antigen-binding site comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 91; and a VL domain comprising the amino acid sequence of SEQ ID NO: 93 or or L) i) The first antigen-binding site comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 74; and a VL domain comprising the amino acid sequence of SEQ ID NO: 75 and ii) The second antigen-binding site comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 90; and a VL domain comprising the amino acid sequence of SEQ ID NO: 93 or or M)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 74; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 90; and VL domain containing the amino acid sequence of SEQ ID NO: 94 including, or N)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 74; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 92; and VL domain containing the amino acid sequence of SEQ ID NO: 93 including, or O)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 74; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 91; and VL domain containing the amino acid sequence of SEQ ID NO: 93 including, or P)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 71; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 92; and VL domain containing the amino acid sequence of SEQ ID NO: 93 It is a bispecific antibody that includes [specific component].

[0020] One embodiment of the present invention is an isolated bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, A)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 71, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I, and (b) X 1 V is X 2 P is X 3 The amino acid sequence GX of sequence number 58 is H. 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site described above is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 90, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 93, and (d)X 1 F is X 2 The amino acid sequence KAX of sequence number 79 is R. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W, and the VL domain sequence including, or B)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 71, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I, and (b) X 1 V is X 2 P is X 3 The amino acid sequence GX of sequence number 58 is H. 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site described above is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 91, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 93, and (d)X 1 F is X 2 The amino acid sequence KAX of sequence number 79 is R. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W, and the VL domain sequence including, or C)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 71, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1is V, I or H, and X 2 is P or H, and X 3 is H or G, the amino acid sequence GX of SEQ ID NO: 58 1 IX 2 IFX 3 a VH domain sequence comprising a CDR-H2 containing TANYAQKFQG, and (c) a CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) a CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) a CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) a CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62 comprising ii) the above second antigen-binding site is a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 90, comprising (a) a CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG where X is D or E, and (c) a CDR-H3 containing the amino acid sequence GVFGFFXH where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 94, comprising (d) X 1 is F or T, and X 2 is R or L, the amino acid sequence KAX of SEQ ID NO: X 1 EDIYNRX 2 A containing CDR-L1, (e) a CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3 containing the amino acid sequence QQFXSAPYT where X is W or R of SEQ ID NO: 81 including, or D)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 72, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site described above is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 90, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 94, and (d)X 1 is F or T, X 2 The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain sequence including, or E)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 73, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site described above is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 90, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 93, and (d)X 1 is F or T, X 2 The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain sequence including, or F)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 73, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site described above is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 90, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 94, and (d)X 1 is F or T, X 2 The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain sequence including, or G)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 73, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site described above is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 92, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 93, and (d)X 1 is F or T, X 2The amino acid sequence KAX of SEQ ID NO: 79, where X is R or L 1 EDIYNRX 2 A CDR-L1 containing A, (e) a CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, the VL domain sequence comprising or H) i) The above first antigen-binding site is a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 73, comprising (a) a CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, (b) X 1 is V, I or H, X 2 is P or H, X 3 is H or G, the amino acid sequence GX 1 IX 2 IFX 3 TANYAQKFQG, and (c) a CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) a CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) a CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) a CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62 comprising ii) The above second antigen-binding site is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 91, comprising: (a) CDR-H1 comprising the amino acid sequence HTYMH of SEQ ID NO: 76; (b) CDR-H2 comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E; and (c) CDR-H3 comprising the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, comprising: (d) CDR-L1 comprising the amino acid sequence KAX 1 EDIYNRX 2 where X is F or T, and X 1 is R or L, and contains A; (e) CDR-L2 comprising the amino acid sequence GATSLEH of SEQ ID NO: 80; and (f) CDR-L3 comprising the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R 2 VL domain sequence comprising or I) i) The first antigen-binding site is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 72, comprising: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T; (b) CDR-H2 comprising the amino acid sequence GX 1 IX 2 IFX 3 where X is V, I or H, X 1 is P or H, and X 2 is H or G, and contains the amino acid sequence TANYAQKFQG; and (c) CDR-H3 comprising the amino acid sequence YDAHYGELDF of SEQ ID NO: 59 3 VH domain sequence; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site described above is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 90, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 93, and (d)X 1 is F or T, X 2 The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain sequence including, or J)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 72, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site described above is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 92, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 93, and (d)X 1 is F or T, X 2The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain sequence including, or K)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 72, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site described above is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 91, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 93, and (d)X 1 is F or T, X 2 The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain sequence including, or L)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 74, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising: (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61; and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. ii) The second antigen-binding site described above is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 90, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 93, and (d)X 1 is F or T, X 2 The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain sequence including, or M)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 74, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site described above is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 90, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 94, and (d)X 1 is F or T, X 2 The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2(e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain sequence including, or N)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 74, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site described above is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 92, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 93, and (d)X 1 is F or T, X 2 The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain sequence including, or O)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 74, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site described above is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 91, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 93, and (d)X 1 is F or T, X 2 The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain sequence including, or P)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 71, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site described above is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 92, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 93, and (d)X 1 is F or T, X 2The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain It is a bispecific antibody that includes [specific component].

[0021] In one embodiment, the bispecific antibody described herein is i) Block the binding of CCL2 to its receptor CCR2 in vitro (reporter assay, IC) 50 =0.5nM); and / or ii) Inhibit CCL2-mediated chemotaxis of myeloid cells in vitro (IC 50 =1.5nM); and / or iii) Cross-reactivity with cynomolgus monkeys and human CCL2.

[0022] In one embodiment, the bispecific antibody described herein does not cross-react with other CCL homologs, and in particular, exhibits 100 times less binding to other CCL homologs (e.g., CCL8) compared to binding to CCL2.

[0023] In one embodiment, the bispecific antibody described herein binds to first and second epitopes on human CCL2 in an ion-dependent manner.

[0024] In one embodiment, the bispecific antibody described herein binds to human CCL2 in a pH-dependent manner, with both the first and second antigen-binding sites binding to CCL2 with higher affinity at neutral pH than at acidic pH.

[0025] In one embodiment, the bispecific antibody described herein binds to human CCL2 at pH 7.4 with 10 times higher affinity than at pH 5.8.

[0026] In one embodiment, the bispecific antibody described herein comprises an IgG1 heavy chain constant domain (or its Fc domain) containing one or more of the following mutations (Kabat EU numbering). i) Q311R and / or P343R (suitable for increasing pI to enhance antigen uptake); and / or ii) L234Y, L235W, G236N, P238D, T250V, V264I, H268D, Q295L, T307P, K326T and / or A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and / or iii) M428L, N434A and / or Y436T (suitable for increasing affinity to FcRn for a longer plasma half-life); and / or iv) Q438R and / or S440E (suitable for inhibiting rheumatoid factor binding).

[0027] In one embodiment, the bispecific antibody described herein comprises an IgG1 heavy chain constant domain (or its Fc domain) containing one or more of the following mutations (Kabat EU numbering). i) Q311R, and / or P343R (suitable for increasing pI to enhance antigen uptake); and / or ii) L235W, G236N, H268D, Q295L, K326T and / or A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and / or iii) N434A (suitable for increasing affinity to FcRn for a longer plasma half-life); and / or iv) Q438R and / or S440E (suitable for inhibiting rheumatoid factor binding).

[0028] In one embodiment, the bispecific antibody described herein comprises an IgG1 heavy chain constant domain (or its Fc domain) containing the following mutation (Kabat EU numbering). i) Q311R and P343R (suitable for increasing pI to enhance antigen uptake); and ii) L235W, G236N, H268D, Q295L, K326T and A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and iii) N434A (suitable for increasing affinity to FcRn for a longer plasma half-life); and iv) Q438R and / S440E (suitable for inhibiting rheumatoid factor binding).

[0029] In one embodiment, the bispecific antibody described herein comprises an IgG1 heavy chain constant domain (or its Fc domain) containing the following mutation (Kabat EU numbering). i) Q311R and P343R (suitable for increasing pI to enhance antigen uptake); and ii) N434A (suitable for increasing affinity to FcRn for a longer plasma half-life); and iii) Q438R and / S440E (suitable for inhibiting rheumatoid factor binding).

[0030] In one embodiment, the bispecific antibody described herein comprises an IgG1 heavy chain constant domain (or its Fc domain) containing the following mutation (Kabat EU numbering).

[0031] Q311R and P343R (suitable for increasing pI to enhance antigen uptake).

[0032] In one embodiment, the bispecific antibody described herein comprises an IgG1 heavy chain constant domain (or its Fc domain) containing one or more of the following mutations (Kabat EU numbering). i) Q311R and / or P343R (suitable for increasing pI to enhance antigen uptake); and / or ii) L234Y, P238D, T250V, V264I, T307P and / or A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and / or iii) M428L, N434A and / or Y436T (suitable for increasing affinity to FcRn for a longer plasma half-life); and / or iv) Q438R and / or S440E (suitable for inhibiting rheumatoid factor binding).

[0033] In one embodiment, the bispecific antibody described herein comprises an IgG1 heavy chain constant domain (or its Fc domain) containing one or more of the following mutations (Kabat EU numbering). i) Q311R and P343R (suitable for increasing pI to enhance antigen uptake); and ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and iii) M428L, N434A and Y436T (suitable for increasing affinity to FcRn for a longer plasma half-life); and iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

[0034] In one embodiment, the bispecific antibody described herein comprises an IgG1 heavy chain constant domain (or its Fc domain) containing one or more of the following mutations (Kabat EU numbering). i) Q311R and P343R (suitable for increasing pI to enhance antigen uptake); and ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and iii) N434A and (suitable for increasing affinity for FcRn due to a longer plasma half-life); and iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

[0035] In one embodiment, the bispecific antibody described herein comprises two IgG1 heavy chain constant domains (or their Fc domains) containing the following mutations (EU numbering) (independently or in addition to the above mutations). i) S354C and T366W in one of the heavy chain constant domains ii) Y349C, T366S, L368A, Y407V in the other heavy chain constant domain

[0036] One embodiment of the present invention is an isolated (monospecific) antibody that binds (specifically) to human CCL2, The antibody is, A) (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain containing CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62, comprising the VL domain. including, or B) VH domains comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and (d)X 1 is F or T, X 2 The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain Includes.

[0037] One embodiment of the present invention is an isolated (monospecific) antibody that binds (specifically) to human CCL2, The antibody is, A) VH domain containing the amino acid sequence of SEQ ID NO: 71; and VL domain containing the amino acid sequence of SEQ ID NO: 75; or B) VH domain containing the amino acid sequence of SEQ ID NO: 72; and VL domain containing the amino acid sequence of SEQ ID NO: 75; or C) VH domain containing the amino acid sequence of SEQ ID NO: 73; and VL domain containing the amino acid sequence of SEQ ID NO: 75; or D) VH domain containing the amino acid sequence of SEQ ID NO: 74; and VL domain containing the amino acid sequence of SEQ ID NO: 75; or E) VH domain containing the amino acid sequence of SEQ ID NO: 90; and VL domain containing the amino acid sequence of SEQ ID NO: 93; or F) VH domain containing the amino acid sequence of SEQ ID NO: 91; and VL domain containing the amino acid sequence of SEQ ID NO: 93; or G) VH domain containing the amino acid sequence of SEQ ID NO: 92; and VL domain containing the amino acid sequence of SEQ ID NO: 93; or H) VH domain containing the amino acid sequence of SEQ ID NO: 90; and VL domain containing the amino acid sequence of SEQ ID NO: 94 Includes.

[0038] One embodiment of the present invention is an isolated nucleic acid encoding a (single or bispecific) antibody described in any one of the prior embodiments.

[0039] One embodiment of the present invention is a host cell containing such nucleic acid.

[0040] One embodiment of the present invention is a method for producing antibodies, comprising culturing such host cells so that antibodies are produced.

[0041] In one embodiment of the present invention, such a method further includes the step of recovering antibodies from host cells.

[0042] One embodiment of the present invention is a pharmaceutical formulation comprising a bispecific antibody and a pharmaceutically acceptable carrier as described herein.

[0043] One embodiment of the present invention is a bispecific antibody described herein for use as a pharmaceutical.

[0044] One embodiment of the present invention is the use of the dual specificity described herein in the manufacture of a pharmaceutical product.

[0045] In one embodiment, such a medicine is for the treatment of cancer.

[0046] In one embodiment, such a pharmaceutical product is for treating inflammatory diseases or autoimmune diseases.

[0047] One embodiment of the present invention is a bispecific antibody described herein for use in the treatment of cancer.

[0048] One embodiment of the present invention is a bispecific antibody described herein for use in the treatment of inflammatory diseases or autoimmune diseases.

[0049] One embodiment of the present invention is a method for treating an individual having cancer, comprising administering to the individual an effective amount of the antibody described herein.

[0050] One embodiment of the present invention is a method for treating an individual having an inflammatory disease or an autoimmune disease, comprising administering to the individual an effective amount of the antibody described herein. [Brief explanation of the drawing]

[0051] [Figure 1] Surface plasmon resonance (Biacore®) sensorgrams showing the binding of monospecific anti-CCL2 antibodies (CNTO888 (=CNTO), 1A5, 1G9, and humanized 11K2 (=11k2)) to recombinant CCL2 and CCL2 homologs. [Figure 2a] The following shows the time course of hCCL2 serum concentrations after IV injection of pre-formed immune complexes. a) Solid line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of monospecific anti-CCL2 antibody CNTO888-SG1 (wild-type IgG1) or b) Dotted line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of monospecific anti-CCL2 antibody CNTO888-SG105 (Fc receptor-binding silencing IgG1) were administered to FcRn transgenic mice. [Figure 2b]The following shows the time course of hCCL2 serum concentrations after IV injection of pre-formed immune complexes. a) Solid line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of monospecific anti-CCL2 antibody 11K2-SG1 (wild-type IgG1) or b) Dotted line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of monospecific anti-CCL2 antibody 11K2-SG105 (Fc receptor-binding silencing IgG1) were administered to FcRn transgenic mice. [Figure 2c] The following shows the time course of hCCL2 serum concentrations after IV injection of pre-formed immune complexes. a) Solid line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of monospecific anti-CCL2 antibody ABN912-SG1 (wild-type IgG1) or b) Dotted line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of monospecific anti-CCL2 antibody ABN912-SG105 (Fc receptor-binding silencing IgG1) were administered to FcRn transgenic mice. [Figure 2d] The following shows the time course of hCCL2 serum concentrations after IV injection of pre-formed immune complexes. a) Solid line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of monospecific anti-CCL2 antibody 1A4-SG1 (wild-type IgG1) or b) Dotted line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of monospecific anti-CCL2 antibody 1A4-SG105 (Fc receptor-binding silencing IgG1) were administered to FcRn transgenic mice. [Figure 2e] The following shows the time course of hCCL2 serum concentrations after IV injection of pre-formed immune complexes. a) Solid line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of monospecific anti-CCL2 antibody 1A5-SG1 (wild-type IgG1) or b) Dotted line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of monospecific anti-CCL2 antibody 1A5-SG105 (Fc receptor-binding silencing IgG1) were administered to FcRn transgenic mice. [Figure 2f]The following shows the time course of hCCL2 serum concentrations after IV injection of pre-formed immune complexes. a) Solid line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of monospecific anti-CCL2 antibody 1G9-SG1 (wild-type IgG1) or b) Dotted line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of monospecific anti-CCL2 antibody 1G9-SG105 (Fc receptor-binding silencing IgG1) were administered to FcRn transgenic mice. [Figure 2g] The following shows the time course of hCCL2 serum concentrations after IV injection of pre-formed immune complexes. a) Solid line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of monospecific anti-CCL2 antibody 2F6-SG1 (wild-type IgG1) or b) Dotted line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of monospecific anti-CCL2 antibody 2F6-SG105 (Fc receptor-binding silencing IgG1) were administered to FcRn transgenic mice. [Figure 3] Figure 3a shows the total serum mouse CCL2 concentration after iv injection, and Figure 3b shows the time course of the antibody-time profile. a) Solid line: 20 mg / kg of monospecific anti-CCL2 antibody 11K2-SG1 (wild-type IgG1) and b) Dotted line: 20 mg / kg of monospecific anti-CCL2 antibody 11K2-SG105 (Fc receptor-binding silencing IgG1) was administered to mice. [Figure 4a] The following shows the time course of hCCL2 serum concentrations after IV injection of pre-formed immune complexes. a) Solid line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of bispecific anti-CCL2 antibody 11K2 / / 1G9-WT IgG1 (wild-type IgG1 with intact Fc receptor binding) or b) Dotted line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of bispecific anti-CCL2 antibody 11K2 / / 1G9-PGLALA (Fc receptor-binding silencing IgG1) were administered to Balb / c mice. [Figure 4b]The following shows the time course of hCCL2 serum concentrations after IV injection of pre-formed immune complexes. a) Solid line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of bispecific anti-CCL2 antibody CNTO888 / / 11K2-WT IgG1 (wild-type IgG1 with intact Fc receptor binding) or b) Dotted line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of bispecific anti-CCL2 antibody CNTO888 / / 11K2-PGLALA (Fc receptor-binding silencing IgG1) were administered to Balb / c mice. [Figure 4c] The following shows the time course of hCCL2 serum concentrations after IV injection of pre-formed immune complexes. a) Solid line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of bispecific anti-CCL2 antibody CNTO888 / / 1G9-WT IgG1 (wild-type IgG1 with intact Fc receptor binding) or b) Dotted line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of bispecific anti-CCL2 antibody 11K2 / / 1G9-PGLALA (Fc receptor-binding silencing IgG1) were administered to Balb / c mice. [Figure 4d] The following shows the time course of hCCL2 serum concentrations after IV injection of pre-formed immune complexes. a) Solid line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of bispecific anti-CCL2 antibody CNTO888 / / 1A5-WT IgG1 (wild-type IgG1 with intact Fc receptor binding) or b) Dotted line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of bispecific anti-CCL2 antibody CNTO888 / / 1A5-PGLALA (Fc receptor-binding silencing IgG1) were administered to Balb / c mice. [Figure 4e]The following shows the time course of hCCL2 serum concentrations after IV injection of pre-formed immune complexes. a) Solid line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of bispecific anti-CCL2 antibody 1A5 / / 1G9-WT IgG1 (wild-type IgG1 with intact Fc receptor binding) or b) Dotted line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of bispecific anti-CCL2 antibody 1A5 / / 1G9-PGLALA (Fc receptor-binding silencing IgG1) were administered to Balb / c mice. [Figure 4f] The following shows the time course of hCCL2 serum concentrations after IV injection of pre-formed immune complexes. a) Solid line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of bispecific anti-CCL2 antibody 11K2 / / 2F6-WT IgG1 (wild-type IgG1 with intact Fc receptor binding) or b) Dotted line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of bispecific anti-CCL2 antibody 11K2 / / 2F6-PGLALA (Fc receptor-binding silencing IgG1) were administered to Balb / c mice. [Figure 4g] The following shows the time course of hCCL2 serum concentrations after IV injection of pre-formed immune complexes. a) Solid line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of bispecific anti-CCL2 antibody ABN912 / / 11K2-WT IgG1 (wild-type IgG1 with intact Fc receptor binding) or b) Dotted line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of bispecific anti-CCL2 antibody ABN912 / / 11K2-PGLALA (Fc receptor-binding silencing IgG1) were administered to Balb / c mice. [Figure 4h]The following shows the time course of hCCL2 serum concentrations after IV injection of pre-formed immune complexes. a) Solid line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of bispecific anti-CCL2 antibody 1A4 / / 2F6-WT IgG1 (wild-type IgG1 with intact Fc receptor binding) or b) Dotted line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of bispecific anti-CCL2 antibody 1A4 / / 2F6-PGLALA (Fc receptor-binding silencing IgG1) were administered to Balb / c mice. [Figure 4i] The following shows the time course of hCCL2 serum concentrations after IV injection of pre-formed immune complexes. a) Solid line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of bispecific anti-CCL2 antibody 1A5 / / 2F6-WT IgG1 (wild-type IgG1 with intact Fc receptor binding) or b) Dotted line: 0.1 mg / kg of human CCL2 (hCCL2) and 20 mg / kg of bispecific anti-CCL2 antibody 1A5 / / 2F6-PGLALA (Fc receptor-binding silencing IgG1) were administered to Balb / c mice. [Figure 5a] Biacore® sensorgram showing the binding profiles of four modified 11K2 and four CNTO888 variant monomer CCL2, as well as 16 bispecific anti-CCL2 antibodies CKLO01-CKLO16 resulting from the combined antigen-binding moieties of each of the four modified 11K2 and four CNTO888 variants, at pH 7.4 (black line) and pH 5.8 (gray line). [Figure 5b] Biacore® sensorgrams showing the binding profiles of four modified 11K2 and four CNTO888 variant monomers of CCL2, and 16 bispecific anti-CCL2 antibodies CKLO01-CKLO16 resulting from the combined antigen-binding moieties of each of the four modified 11K2 and four CNTO888 variants. A further dissociation phase at pH 5.8 was incorporated into the BIACORE® assay immediately after the dissociation phase at pH 7.4. [Figure 6]Biacore® sensorgram showing the binding profiles of bispecific anti-CCL2 antibodies CKLO01, CKLO02, CKLO03, and CKLO04 to monomeric CCL8 at pH 7.4 (black line) and pH 5.8 (gray line). [Figure 7a] Serum concentrations of hCCL2 over time after injection of a pre-formed immune complex consisting of hCCL2 and bispecific anti-CCL2 antibodies (parent CNTO / / 11K2 and pH-dependent variants CKLO01, CKLO02, CKLO03, and CKLO04) into SCID mice. [Figure 7b] Serum concentrations of hCCL2 over time after injection of a pre-formed immune complex consisting of hCCL2 and CKLO03 (containing IgG1 wild-type Fc) or CKLO03-SG1099 (CKLO03 with enhanced pI Fc) into SCID mice. [Figure 8] Chemotaxis assay: Bispecific anti-CCL2 antibodies having the same CDR and variable region VH / VL (i.e., CKLO2-IgG1 wild-type and CKLO2-SG1095) but different Fc regions can inhibit THP-1 cell migration with the same potency (IC50 = 0.2 μg / ml; Figure 8, left panel). Similarly, pH-independent CCL2-0048 (unmodified bispecific antibody CNTO888 / 11k2k2 IgG1, the parent of CKLO2) also shows IC50 values ​​of 0 and 2 μg / ml, as pH dependence is important for antigen sweeping (a phenomenon that does not occur in this assay). The corresponding monospecific antibodies CNTO888 IgG1 and humanized 11k2 IgG1 show IC50 values ​​of 0.3 and 0.7 μg / ml, respectively, but the huIgG1 isotype control shows no inhibition (Figure 8, right panel). [Figure 9] In vivo antitumor activity in genetically modified mouse models. Treatment of mouse tumor models with Mab CKLO2-IgG1 (Fc wild-type IgG1) and CKLO2-SG1099 (=CKLO2 pI enhancement). Tumor volume (left), tumor weight (center), and M-MDSC infiltration (right) at the end of the study. (Black represents vehicle, gray represents CKLO2 wild-type IgG1, and white bars / dotted lines represent CKLO2 pI enhancement Fc (CKLO2-SG1099)). [Figure 10] Serum total (left) and free (right) CCL2 levels during in vivo antitumor activity tests (see efficacy in Figure 9) treated with bispecific anti-CCL2 antibody (black for vehicle, gray for CKLO2 wild-type IgG1, and white bars / dotted lines for pI-enhanced Fc (CKLO2-SG1099)). [Figure 11] Proof-of-concept study of CCL2 sweep efficiency in cynomolgus monkeys. Total antibody concentration-time profiles in serum of cynomolgus monkeys; left panel: mean concentration-time profiles of four antibodies over 7 days; Group 1: monospecific CNTO888-SG1 (=IgG1 wild-type) anti-CCL2 antibody as a control for maximum total CCL2 accumulation (n=3 animals); Group 2: biparatopic anti-CCL2 antibody CKLO2-SG1 (IgG1 wild-type) with pH-dependent target binding but without Fc modification (n=3); Group 3: biparatopic anti-CCL2 antibody CKLO2-SG1100 with pH-dependent target binding, Fc-pI and further modifications (n=4); and Group 4: biparatopic anti-CCL2 antibody CKLO2-SG1095 with pH-dependent target binding, Fc-pI and FcγRIIb affinity enhancement and further modifications (n=4). Right panel: Shows the individual concentration-time profiles of individual 4 (group 2) over the PK test period (70 days). [Figure 12] Proof-of-concept study of CCL2 sweep efficiency in cynomolgus monkeys. Total CCL2 concentration-time profiles in serum of cynomolgus monkeys; left panel: average total CCL2 concentration-time profiles of four antibodies over 7 days; right panel: individual total CCL2 concentration-time profiles of 4 individuals (2 groups) over the PK study period (70 days). [Figure 13] Serum free CCL2 concentration-time profiles of cynomolgus monkeys; left panel: average free CCL2 concentration-time profiles of four antibodies over 7 days; right panel: individual free CCL2 concentration-time profiles of 4 individuals (group 2) over the PK test period (70 days); for samples below the detection limit, the average profile was calculated using a value of 0.01 ng / mL (limit of quantification). [Figure 14]PK / PD study of CCL2 sweep efficiency in cynomolgus monkeys. Total CKL02-SG1095 concentration-time profiles in serum of cynomolgus monkeys (CKL02-SG1095 treatment at different concentrations (groups 1-3)); left panel: mean concentration-time profiles (n=4) for three dose levels over 7 days; right panel: individual concentration-time profiles of two ADA-negative individuals (25 mg / kg dose group) over the study period (98 days). [Figure 15] PK / PD study of CCL2 sweep efficiency in cynomolgus monkeys. Comparison of total CCL2 concentration-time profiles in serum of cynomolgus monkeys treated with CKL02-SG1095 at different concentrations (groups 1-3) and those treated with CNTO888-SG1 (group 4); Left panel: Mean total CCL2 concentration-time profiles of the four test groups (error bars indicate SD) over 7 days; Right panel: Individual total CCL2 concentration-time profiles of ADA-negative animals in group 3 (n=2, error bars indicate range) and group 4 (n=3, error bars indicate SD) over the PK study period (98 days). [Figure 16] PK / PD study of CCL2 sweep efficiency in cynomolgus monkeys. Serum free CCL2 concentration-time profiles of cynomolgus monkeys; left panel: mean free CCL2 concentration-time profiles of four test groups (error bars indicate SD) over 7 days (comparison with CKL02-SG1095 treatment (groups 1-3) and CNTO888-SG1 treatment (group 4) at different concentrations); right panel: mean free CCL2 concentration-time profiles of ADA-negative animals in groups 3 (n=2, error bars indicate range) and 4 (n=3, error bars indicate SD) over the PK study period (70 days); mean profiles were calculated using a value of 0.01 ng / mL (limit of quantification) for samples below the detection limit. [Modes for carrying out the invention]

[0052] Detailed description of the invention The present invention relates to a bispecific anti-CCL2 antibody that binds to two different epitopes on human CCL2, a pharmaceutical composition thereof, the manufacture thereof, and its use as a pharmaceutical for the treatment of cancer, inflammatory diseases, autoimmune diseases, and ophthalmic diseases. Accordingly, the antibody comprises a first antigen-binding site that (specifically) binds to a first epitope on human CC2, and a different second antigen-binding site that (specifically) binds to a different second epitope.

[0053] The present invention includes a bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2. i) The first antigen-binding site described above is A VH domain comprising the amino acid sequence of SEQ ID NO: 39, wherein (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 33, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 34, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 35; and A VL domain comprising the amino acid sequence of SEQ ID NO: 40, wherein (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 36, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 37, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 38. The antibody containing the antibody binds to the same epitope on CCL2, ii) The second antigen-binding site described above is A VH domain comprising the amino acid sequence of SEQ ID NO: 47, wherein (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 41, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 43; and A VL domain comprising the amino acid sequence of SEQ ID NO: 48, wherein (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 44, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46. It binds to the same epitope on CCL2 as the antibody containing it.

[0054] In one embodiment, the in vivo clearance rate (ml / day / kg) of human CCL2 after administration of a bispecific antibody containing the constant heavy chain domain (or its Fc domain) of a human wild-type IgG1 isotype was at least 15 times higher, and particularly at least 20 times higher, when a pre-formed immune complex consisting of 20 mg / kg of each bispecific antibody and 0.1 mg / kg of human CCL2 was administered to FcRn transgenic mice in a single dose of 10 ml / kg, compared to the in vivo clearance rate (ml / day / kg) of human CCL2 after administration of a bispecific antibody containing the constant heavy chain domain (or its Fc domain) of a human IgG1 isotype including mutant L234A, L235A, and P329G (Kabat EU numbering).

[0055] The term "epitope" includes any polypeptide determinant that can specifically bind to an antibody. In certain embodiments, the epitope determinant includes a chemically active surface group of a molecule, such as amino acids, sugar side chains, phosphoryls, or sulfonyls, and in certain embodiments, may have specific three-dimensional structural features and / or specific charge features. An epitope is a region of an antigen to which an antibody binds. The present invention relates to a bispecific anti-CCL2 antibody that binds to two different epitopes on human CCL2. The terms first epitope and second epitope refer to two different epitopes on human CCL2. Thus, the second epitope is distinct from the first epitope. Whether an antibody binds to the same epitope as the reference anti-CCL2 antigen binding site, or whether there is competition for binding, can be readily determined by using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope as the reference anti-CCL2 antigen binding site of the present invention, the reference antibody is bound to its CCL2 domain under saturated conditions. Next, the ability of the test antibody to bind to human CCL2 is evaluated. If the test antibody can bind to human CCL2 after saturated binding to the reference anti-CCL2 antigen binding site, it can be concluded that the test antibody binds to an epitope different from the reference anti-CCL2 antigen binding site. On the other hand, if the test antibody cannot bind to human CCL2 after saturated binding to the reference anti-CCL2 antibody, the test antibody may bind to the same epitope as the one bound by the reference anti-CCL2 antibody of the present invention. Further routine experiments (e.g., peptide mutation and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody, or whether steric barrier (or another phenomenon) is the cause of the observed lack of binding. These types of experiments can be performed using ELISA, RIA, surface plasmon resonance (e.g., Biacore), flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.According to certain embodiments of the present invention, two antibodies bind to the same (or overlapping) epitopes if, for example, one antibody in a 1x, 5x, 10x, 20x, or 100x excess inhibits the binding of the other antibody by at least 50%, preferably 75%, 90%, or even 99%, as measured by a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502).

[0056] Alternatively, if substantially all of the amino acid mutations of an antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody, then the two antibodies are considered to bind to the same epitope. Two antibodies are considered to have a "duplicate epitope" if only a subset of the amino acid mutations that reduce or eliminate the binding of one antibody also reduces or eliminates the binding of the other antibody.

[0057] To determine whether an antibody competes with a reference anti-CCL2 antibody for binding, the above binding methodology is performed in two directions. In the first direction, the reference antibody is bound to CCL2 under saturated conditions, and then the binding of the test antibody to human CCL2 is evaluated. In the second direction, the test antibody is bound to the CCL2 molecule under saturated conditions, and then the binding of the reference antibody to human CCL2 is evaluated. In both directions, if only the first (saturated) antibody can bind to the CCL2 molecule, it is concluded that the test antibody and the reference antibody compete for binding to CCL2. As will be understood by those skilled in the art, an antibody that competes with a reference antibody for binding does not necessarily bind to the same epitope as the reference antibody, but can sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0058] As used herein, the terms "CCL2," also called "MCP-1," and "human CCL2" refer to the 76-amino acid sequence variously known as CCL2, MCP-1 (monocyte chemotactic protein 1), SMC-CF (smooth muscle cell chemotactic factor), LDCF (lymphocyte-derived chemotactic factor), GDCF (glioma-derived monocyte chemotactic factor), TDCF (tumor-derived chemotactic factor), HCl1 (human cytokine 11), and MCAF (monocyte chemotactic and activating factor). The gene symbol is SCYA2, the JE gene on human chromosome 17, and the new name is CCL2 (Zlotnik, Yoshie 2000. Immunity 12:121-127). JE is the mouse homolog of human MCP-1 / CCL2.

[0059] Handel et al. (Biochemistry. 1996;35:6569-6584) determined the solution structure of the CCL2 dimer. These studies showed that the secondary structure of CCL2 consists of four β-sheets. Furthermore, the residues responsible for the dimerization interface of CCL2 were described by Zhang and Rollins (Mol Cell Biol. 1995;15:4851-4855). The protein complex appears elongated, oriented so that the two monomers form a large pocket. The structures of the monomeric and dimeric CCL2 in two crystalline forms, the so-called I and P forms, have also been determined (Lubkowski et al., Nat Struct Biol. 1997;4:64-69). Paolini et al. (J Immunol. 1994 Sep 15;153(6):2704-17) described the presence of MCP1 / CCL2 as monomers at physiologically appropriate concentrations:rec. By analyzing CCL2 protein (purchased from Peprotech) using size exclusion HPLC, sedimentation equilibrium ultracentrifugation, and chemical crosslinking, we were able to demonstrate that the weight fractions of monomeric and dimeric MCP-1 depend on in vitro cooperation. Finally, Seo and collaborators (J Am Chem Soc. 2013 Mar 20;135(11):4325-32) were able to demonstrate the presence of injected CCL2 in both monomeric and dimeric forms under physiological conditions by ion mobility mass spectrometry.

[0060] Therefore, while "wild-type CCL-2" (wt CCL2) can exist as a monomer, it can also actually form dimers at physiological concentrations. This monomer-dimer equilibrium is indeed different and must be carefully considered in all described in vitro experiments where different concentrations may be used. To avoid uncertainty, we have created a point-mutation CCL2 variant: the "P8A" variant of CCL2 harbors a mutation at the dimerization interface and, as a result, cannot form a dimer that yields the defined pure CCL2 monomer. In contrast, the "T10C" variant of CCL2 yields a fixed dimer of CCL2 (J Am Chem Soc. 2013 Mar 20;135(11):4325-32).

[0061] The CCL2 / CCR2 axis is the primary mediator of the recruitment of immature myeloid cells to tumors. CCL2 is overexpressed by malignant cells and binds to the extracellular matrix (ECM), which constructs a chemoattractant gradient. Once in the tumor, myeloid suppressor cells (MDSCs) contribute to a protumorogenic environment by secreting / upregulating anti-inflammatory cytokines / receptors that inhibit the initiation of antitumor T cell responses. In this way, MDSCs may reduce or even impair the effectiveness of any T cell activation therapy (Meyer et al, 2014). Therefore, specific inhibition of the recruitment of these immature myeloid cells enhances the effectiveness of checkpoint inhibitors, T cell bispecificity, and cancer immunotherapy. Furthermore, CCL2 is also involved in promoting angiogenesis, metastasis, and tumor growth, suggesting that neutralizing CCL2 may contribute to several antitumor interventions.

[0062] Targeting CCL2, in contrast to its receptor, specifically inhibits undesirable CCL2-mediated effects and saves signaling through the same receptor (CCL2) but different ligands (e.g., CCL7, CCL8, CCL13) that are involved in the recruitment of other immune cell populations such as Th1 and NK cells.

[0063] Clinically, CCL2 has been a preferred antibody target in several studies aimed at neutralizing its elevated levels caused by various inflammatory diseases such as rheumatoid arthritis (Haringman et al, 2006), idiopathic pulmonary fibrosis (Raghu et al, 2015), diabetic nephropathy (Menne et al, 2016), and cancer (Sandhu et al, 2013). However, its high synthesis rate, along with the observed high in vivo antibody-antigen dissociation constant (KD), has proven to be a major obstacle preventing the suppression of free CCL2 by conventional antibodies at clinically viable doses (Fetterly et al, 2013).

[0064] CCL2 neutralization has been observed in several types of cancer, including breast cancer (BC), ovarian cancer (OvCa), colorectal cancer (CRC), pancreatic cancer, and prostate cancer, and appears to be more clearly associated with elevated serum levels of CCL2 in patients. However, even in patients within these indications, where the tumor is highly infiltrated by myeloid immune cells, without presenting this serological evidence, patients can still greatly benefit from this novel therapy due to the many roles that CCL2 plays in the tumor situation, as described above.

[0065] As used herein, an antibody that "binds to human CCL2," "specifically binds to human CCL2," "binds to human CCL2," or is an "anti-CCL2" antibody is defined as 5.0 × 10⁻⁶. -8 K below mol / L D Value, in one embodiment, 1.0 × 10 -9 K below mol / L D Value, in one embodiment, 5.0 × 10 -8 mol / L ~ 1.0 × 10 -13 K in mol / l D This refers to antibodies that specifically bind to the human CCL2 antigen based on their binding affinity.

[0066] Binding affinity is determined by a standard binding assay, such as surface plasmon resonance technology (BIAcore®, GE-Healthcare Uppsala, Sweden), using a construct containing, for example, the extracellular domain of CCL2 (e.g., its naturally occurring three-dimensional structure). In one embodiment, binding affinity is determined by a standard binding assay using exemplary soluble CCL2.

[0067] Antibody specificity refers to the selective recognition of an antibody against a specific epitope of an antigen. For example, naturally occurring antibodies are monospecific.

[0068] As used herein, the term “single-specific” antibody refers to an antibody that has one or more binding sites, each binding to the same epitope of the same antigen.

[0069] As used herein, the terms “(human) CCL2-binding bispecific antibody,” “(human) CCL2-binding biparatopic antibody,” “bispecific anti-CCL2 antibody,” and “biparatopic anti-CCL2 antibody” mean that the antibody can specifically bind to at least two different epitopes on (human) CCL2. Typically, such a bispecific antibody contains two different antigen-binding sites (two different paratopes), each specific to a different epitope on (human) CCL2. In certain embodiments, the bispecific antibody can bind to two different, non-overlapping epitopes on CCL2, meaning that the two different antigen-binding sites do not compete for binding to CCL2.

[0070] For the purposes of this specification, “receptor human framework” means a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. A human immunoglobulin framework or human consensus framework “derived” acceptor human framework may contain the same amino acid sequence or may contain a modification of the amino acid sequence. In some embodiments, the number of amino acid modifications is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is sequence-identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0071] The term "antibody" is used here in its broadest sense, encompassing a variety of antibody structures, including (but not limited to) monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0072] An "antibody fragment" refers to a molecule other than an intact antibody, which contains a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0073] As used herein, the term "valency" refers to the presence of a specific number of antigen-binding sites within an antibody. Therefore, the term "monovalent binding to an antigen" refers to the presence of one (and not more than one) antigen-specific antigen-binding sites within the antibody.

[0074] The term “antigen-binding site” refers to a site or region of an antibody that provides interaction with an antigen, i.e., one or more amino acid residues. For example, the antigen-binding site of an antibody includes amino acid residues from the complementarity-determining region (CDR). In one embodiment, the antigen-binding site of an antibody includes amino acid residues derived from VH and VL. Native immunoglobulin molecules typically contain two antigen-binding sites; Fab molecules typically have one antigen-binding site. “Antigen-binding moiety” refers to a polypeptide molecule that contains an antigen-binding site that specifically binds to an antigenic determinant. Antigen-binding moieties include antibodies and fragments thereof as further defined herein. A particular antigen-binding moiety includes the antigen-binding domain of an antibody, which includes an antibody heavy chain variable region and an antibody light chain variable region. In a particular embodiment, the antigen-binding moiety may include an antibody constant region as further defined herein and known in the art. Useful heavy chain constant regions include any of five isotypes: α, δ, ε, γ, or μ. Useful light chain constant regions include any of two isotypes: κ and λ.

[0075] As used herein, the terms “antigenic determinant” or “antigen” refer to a site on a polypeptide polymer to which an antigen-binding moiety / site binds to form an antigen-binding moiety-antigen complex. Useful antigenic determinants may be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM).

[0076] The term "chimeric" antibody refers to an antibody in which part of the heavy chain and / or light chain originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0077] The “class” of an antibody refers to the type of constant domain or constant region held by its heavy chain. Antibodies have five main classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulin are called α, δ, ε, γ, and μ, respectively. Preferably, the bispecific antibodies of the present invention are human IgG isotypes, and more preferably human IgG1 isotypes. As used herein, the terms IgG isotype and IgG1 isotype refer to human IgG isotype and human IgG1 isotype. Typically, different IgG isotypes exist in the form of slightly different allotypes based on allelic mutations between IgG subclasses (see Vidarsson et al.; Front Immunol 5 (2014) Article 520, 1-17). The "effective dose" of a drug, such as a pharmaceutical preparation, refers to the amount that is effective in the dosage and duration required to achieve the desired therapeutic or preventive outcome.

[0078] In this specification, the terms “Fc domain” or “Fc region” are used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes both the native sequence Fc region and the variant Fc region. While the boundaries of the Fc region of an IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is typically defined to extend from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more amino acids, particularly one or two, from the C-terminus of the heavy chain. Thus, by expression of a particular nucleic acid molecule encoding a full-length heavy chain, antibodies produced by host cells may contain the full-length heavy chain or a cleaved variant of the full-length heavy chain (also referred to herein as a “cleaved variant heavy chain”). This may be the case where the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbered according to the Kabat EU index). Therefore, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (K447) of the Fc domain may or may not be present. Unless otherwise specified, the amino acid sequence of the heavy chain containing the Fc domain (or a subunit of the Fc domain as defined herein) is shown herein without the C-terminal glycine-lysine dipeptide. In one embodiment of the present invention, the heavy chain containing the Fc domain subunit specified herein in the antibody or bispecific antibody according to the present invention includes a further C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In another embodiment of the present invention, the heavy chain containing the Fc domain subunit specified herein in the antibody or bispecific antibody according to the present invention includes a further C-terminal glycine residue (G446, numbered according to the Kabat EU index). A composition of the present invention, for example, a pharmaceutical composition described herein, comprises a collection of the antibody or bispecific antibody of the present invention. The group of antibodies or bispecific antibodies may include molecules having full-length heavy chains and molecules having cleaved variant heavy chains.The antibody or bispecific antibody collection may consist of a mixture of molecules having full-length heavy chains and molecules having cleaved variant heavy chains, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the antibody or bispecific antibody has cleaved variant heavy chains. In one embodiment of the present invention, a composition comprising the antibody or bispecific antibody collection of the present invention comprises an antibody or bispecific antibody comprising a heavy chain containing a subunit of the Fc domain as specified herein, comprising a further C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to Kabat's EU index). In one embodiment of the present invention, a composition comprising the antibody or bispecific antibody collection of the present invention comprises an antibody or bispecific antibody comprising a heavy chain containing a subunit of the Fc domain as specified herein, comprising a further C-terminal glycine residue (G446, numbered according to Kabat's EU index). In one embodiment of the present invention, such a composition comprises an antibody or a collection of bispecific antibodies comprising a molecule comprising a heavy chain comprising a subunit of the Fc domain as specified herein, a molecule comprising a heavy chain comprising a subunit of the Fc domain as specified herein, comprising a further C-terminal glycine residue (G446, numbered according to the Kabat EU Index), and a molecule comprising a heavy chain comprising a subunit of the Fc domain as specified herein, comprising a further C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU Index). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the “EU numbering system” (also called “Kabat EU numbering” or “Kabat EU numbering”) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above).As used herein, the term "subunit" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide that includes the C-terminal constant region of an immunoglobulin heavy chain and possesses stable self-associating ability. For example, a subunit of an IgG Fc domain includes the IgG CH2 and IgG CH3 constant domains.

[0079] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the CDR and FR sequences generally appear in VH (or VL) in the following sequence: FR-H1(L1)-CDR-H1(L1)-FR-H2(L2)-CDR-H2(L2)-FR-H3(L3)-CDR-H3(L3)-FR-H4(L4).

[0080] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably in this specification and refer to antibodies that have a structure substantially similar to that of a natural antibody or that have a heavy chain containing an Fc region as defined herein.

[0081] A "human antibody" is defined as an antibody produced by a human or human cell, or an antibody that has an amino acid sequence corresponding to the amino acids of an antibody of non-human origin that utilizes a human antibody repertoire or a sequence encoding a human antibody. This definition of a human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues.

[0082] The "Human Consensus Framework" is a framework representing the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from subgroups of variable domain sequences. Generally, the sequence subgroups are those described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Bethesda MD (1991), NIH Publication 91-3242, Vols. 1-3. In one embodiment, with respect to VL, the subgroup is subgroup Kappa I, as described by Kabat et al. (see above). In one embodiment, with respect to VH, the subgroup is subgroup III, as described by Kabat et al. (see above).

[0083] A “humanized” antibody refers to a chimeric antibody containing amino acid residues derived from a non-human CDR and amino acid residues derived from a human FR. In certain embodiments, the humanized antibody contains substantially all of at least one, typically two, variable domains, where all or substantially all of the CDR corresponds to that of a non-human antibody, and all or substantially all of the FR corresponds to that of a human antibody. The humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0084] The terms “complementary chain determination region” or “CDR,” as used herein, refer to each region of an antibody variable domain that is hypervariable in sequence and / or structurally forms a predetermined loop (“hypervariable loop”) and / or contains residues that contact an antigen (“antigen contact”). Generally, an antibody contains six CDRs, three located in the VH (CDR-H1, CDR-H2, CDR-H3) and three located in the VL (CDR-L1, CDR-L2, CDR-L3). Examples of CDRs as used herein include: (a) Hypervariable loops formed at amino acid residues 26-32 (CDR-L1), 50-52 (CDR-L2), 91-96 (CDR-L3), 26-32 (CDR-H1), 53-55 (CDR-H2), and 96-101 (CDR-H3) (Chothia and Lesk, J.Mol.Biol.196:901-917(1987)); (b) CDRs generated at amino acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), 89-97 (CDR-L3), 31-35b (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contact occurring at amino acid residues 27c-36 (CDR-L1), 46-55 (CDR-L2), 89-96 (CDR-L3), 30-35b (CDR-H1), 47-58 (CDR-H2), and 93-101 (CDR-H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)); and (d) A combination of (a), (b), and / or (c) containing CDR amino acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), 89-97 (vL3), 31-35 (CDR-H1), 50-63 (CDR-H2), and 95-102 (CDR-H3).

[0085] Unless otherwise indicated, CDR residues and other residues (e.g., FR residues) in variable domains are numbered herein according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0086] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, e.g., monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0087] An “isolated” antibody is one that has been separated from its natural environment. In some embodiments, antibodies are purified to a purity of over 95% or over 99%, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman, S. et al., J. Chromatogr. B 848 (2007) 79-87.

[0088] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from their natural environment. Isolated nucleic acids include nucleic acid molecules that are normally found inside cells containing nucleic acid molecules, but the nucleic acid molecules are located outside of chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0089] "Isolated nucleic acids encoding a single or bispecific anti-CCL2 antibody" means one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, including such nucleic acid molecules(s) in a single vector or separate vectors, and such nucleic acid molecules(s) are located at one or more locations within a host cell.

[0090] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting that population are identical and / or bind to the same epitope, with the exception of possible variant antibodies, such as those containing spontaneous mutations or arising during the production of a monoclonal antibody preparation, which are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed toward different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed toward a single determinant on one antigen. Therefore, the modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous collection of antibodies and should not be interpreted as requiring antibody production by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced by a variety of techniques, including (but not limited to) hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such and other exemplary methods for producing monoclonal antibodies are described herein.

[0091] A "native antibody" refers to a native immunoglobulin molecule with a variety of structures. For example, a natural IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons, containing two identical light chains and two identical heavy chains linked by disulfide bonds. Each heavy chain from the N-terminus to the C-terminus has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, each light chain from the N-terminus to the C-terminus has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be assigned to one of two types called kappa (κ) or lambda (λ).

[0092] The term “package insert” is used to refer to the instructions typically included on the market packaging of a therapeutic product, including information relating to indications, use, dosage, administration, combination therapy, contraindications, and / or warnings for such therapeutic products.

[0093] The "amino acid sequence identity percentage (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps if necessary to achieve the maximum sequence identity percentage, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the amino acid sequence identity percentage can be achieved in various methods within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared. However, for the purposes of this specification, the amino acid sequence identity % values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code, along with user documentation, has been filed with the U.S. Copyright Office, Washington DC, 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California) or can be compiled from its source code. The ALIGN-2 program should be compiled for use with UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.

[0094] In situations where ALIGN-2 is used for amino acid sequence comparison, the amino acid sequence identity percentage of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (or, a given amino acid sequence A may be described as having or containing a certain amino acid sequence identity percentage to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y In the formula, X is the number of amino acid residues scored as identity matches by the sequence alignment program ALIGN-2 in the alignment of A and B in this program, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity % of A to B is not equal to the amino acid sequence identity % of B to A. Unless otherwise specifically stated, all amino acid sequence identity % values ​​used herein are obtained as described in the paragraph immediately preceding the use of the ALIGN-2 computer program.

[0095] The term "pharmaceutical preparation" refers to a preparation in which the biological activity of the active ingredient contained therein is effective, and which does not contain any additional components that are unacceptably toxic to the person to whom the preparation is administered.

[0096] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical preparation other than the active ingredient that is non-toxic to the target substance. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0097] As used herein, “treatment” (and its grammatical variations, e.g., “to treat” or “to treat”) refers to a clinical intervention in an attempt to alter the natural course of the treated individual, and may be carried out for preventive purposes or during the course of a clinicopathological condition. Desired effects of treatment include preventing the onset or recurrence of the disease, reducing symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, achieving remission or mitigation of the condition, and achieving a recovered or improved prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of the disease or to slow the progression of the disease.

[0098] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in antibody-antigen binding. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (CDRs). (See, for example, Kindt, T.Jet al., Kuby Immunology, 6th ed., WH Freeman and Co., NY (2007), p. 91.) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by screening a library of complementary VL or VH domains, respectively, using the VH or VL domain of the antibody that binds to the antigen. For example, see Portolano, S. et al., J.Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

[0099] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures, and vectors incorporated into the genome of a host cell into which they are introduced. Certain vectors can direct the expression of the nucleic acid to which they are functionally linked. Such vectors are referred to herein as "expression vectors."

[0100] I. Compositions and Methods In one embodiment, the present invention is partially based on the finding that the bispecific antibodies described herein utilize different anti-CCL2 antigen-binding sites as first and second antigen-binding sites / parts. These anti-CCL2 antibodies have the ability to bind with high specificity to specific epitopes of CCL2 and to specifically inhibit the binding of CCL2 to its receptor CCL2. They exhibit improved immune complex formation and improved CCL2 suppression in vivo compared to monospecific antibodies.

[0101] Bispecific anti-CCL2 antibody bispecific antibody The bispecific antibodies described herein are monoclonal antibodies having binding specificity to at least two different epitopes on CCL2.

[0102] Techniques for producing multispecific and bispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)) and "knob-in-hole" operations (see, e.g., U.S. Patent No. 5,731,168 and Atwell et al., J.Mol.Biol.270:26 (1997)). Multispecific antibodies are also produced by manipulating the electrostatic steering effect to create antibody Fc heterodimer molecules (see, e.g., International Publication No. 2009 / 089004); crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); producing bispecific antibodies using leucine zippers (see, e.g., Kostelny et al., J.Immunol., 148(5):1547-1553 (1992) and International Publication No. 2011 / 034605); using common light chain techniques to avoid light chain mispairing problems (see, e.g., International Publication No. 98 / 50431); and using "diabody" techniques to produce bispecific antibody fragments (see, e.g., Hollinger et al.) It can be prepared by using single-stranded Fv(sFv) dimers (see, for example, Gruber et al., J.Immunol., 152:5368 (1994)); and by preparing a trispecific antibody as described, for example, Tutt et al. J.Immunol. 147:60 (1991).

[0103] For example, this also includes manipulated antibodies having three or more antigen-binding sites, such as "octopus antibodies," or DVD-Ig (see, for example, International Publications 2001 / 77342 and 2008 / 024715). Other examples of multispecific antibodies having three or more antigen-binding sites can be found in International Publications 2010 / 115589, 2010 / 112193, 2010 / 136172, 2010 / 145792, and 2013 / 026831. Bispecific antibodies or their antigen-binding fragments also include "dual-acting FAbs" or "DAFs" that contain antigen-binding sites that bind to CCL2 and another different antigen, or to two different epitopes of CCL2 (see, for example, U.S. Patent Application Publication 2008 / 0069820 and International Publication 2015 / 095539).

[0104] Multispecific antibodies can also be provided in an asymmetric form having domain crossovers on one or more binding arms of the same antigen specificity, i.e., by exchanging VH / VL domains (see, e.g., International Publication Nos. 2009 / 080252 and 2015 / 150447), CH1 / CL domains (see, e.g., International Publication No. 2009 / 080253), or complete Fab arms (see, e.g., International Publication Nos. 2009 / 080251 and 2016 / 016299, also see Schaefer et al., PNAS, 108(2011)1187-1191, and Klein et al., MAbs 8(2016)1010-20) (also known as CrossMab). Asymmetric binding arms can also be manipulated by introducing charged or uncharged amino acid mutations at the domain interface to direct correct Fab pairings. For example, see International Publication No. 2016 / 172485.

[0105] Various further molecular formats of multispecific antibodies are known in the art and are included herein (see, for example, Spiess et al., Mol Immunol 67(2015) 95-106).

[0106] Preferred bispecific antibody format. According to certain embodiments of the present invention, the bispecific antibodies described herein have domain crossover by exchanging one or more binding arms of the same antigen specificity with a VH / VL domain (see, for example, International Publication No. 2009 / 080252 and International Publication No. 2015 / 150447), a CH1 / CL domain (see International Publication No. 2009 / 080253), or a complete Fab arm (see International Publication No. 2009 / 080251, International Publication No. 2016 / 016299, Schaefer et al., PNAS, 108(2011)1187-1191, and Klein et al., MAbs 8(2016)1010-20).

[0107] Such bispecific antibodies may involve charge modification, particularly VH / VL domain exchange (see International Publication No. 2015 / 150447): The bispecific antibodies of the present invention may include amino acid substitutions in the Fab molecules contained herein that are particularly efficient in reducing mispairing of the light chain with an incompatible heavy chain (Bence-Jones type byproduct) that can occur in the production of Fab-based bispecific antibodies / antibodies having VH / VL exchange in one (or more, in the case of molecules containing more than two antigen-binding Fab molecules) binding arms (see International Publication No. 2015 / 150447, which is incorporated herein by reference in its entirety, and especially the examples contained therein). The ratio of the desired bispecific antibody to undesirable byproducts, particularly Bence-Jones type byproducts that occur in bispecific antibodies having VH / VL domain exchange in one of the binding arms, can be improved by introducing charged amino acids having the opposite charge to specific amino acid positions in the CH1 and CL domains (sometimes referred to herein as "charge modification").

[0108] Therefore, in some embodiments, both the first and second antigen-binding moieties of a bispecific antibody are Fab molecules, and in one of the antigen-binding moieties (particularly the second antigen-binding moiety), the variable domains VL and VH of the Fab light chain and Fab heavy chain are interchangeable. i) In the constant domain CL of the first antigen-binding moiety, the amino acid at position 124 is substituted with a positively charged amino acid (Kabat numbering), and in the constant domain CH1 of the first antigen-binding moiety, the amino acid at position 147 or the amino acid at position 213 is substituted with a negatively charged amino acid (Kabat EU index numbering), or ii) In the constant domain CL of the second antigen-binding region, the amino acid at position 124 is substituted with a positively charged amino acid (Kabat numbering), and in the constant domain CH1 of the second antigen-binding region, the amino acid at position 147 or the amino acid at position 213 is substituted with a negatively charged amino acid (Kabat EU index numbering).

[0109] Bispecific antibodies do not contain both of the modifications described in i) and ii). The constant domains CL and CH1 of the antigen-binding moiety where VH / VL are replaced are not replaced by each other (i.e., remain unexchanged).

[0110] In a more specific embodiment, i) In the constant domain CL of the first antigen-binding moiety, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering), and in the constant domain CH1 of the first antigen-binding moiety, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering), or ii) In the constant domain CL of the second antigen-binding moiety, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering), and in the constant domain CH1 of the second antigen-binding moiety, the amino acid at position 147 or position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0111] In one such embodiment, in the constant domain CL of the first antigen-binding moiety, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering), and in the constant domain CH1 of the first antigen-binding moiety, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0112] In further embodiments, in the constant domain CL of the first antigen-binding moiety, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering), and in the constant domain CH1 of the first antigen-binding moiety, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0113] In certain embodiments, in the constant domain CL of the first antigen-binding moiety, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering), the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering), in the constant domain CH1 of the first antigen-binding moiety, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0114] In a further specific embodiment, in the constant domain CL of the first antigen-binding moiety, the amino acid at position 124 is substituted with lysine (K) (Kabat numbering), the amino acid at position 123 is substituted with lysine (K) (Kabat numbering), in the constant domain CH1 of the first antigen-binding moiety, the amino acid at position 147 is substituted with glutamic acid (E) (Kabat EU index numbering), and the amino acid at position 213 is substituted with glutamic acid (E) (Kabat EU index numbering).

[0115] In a further specific embodiment, in the constant domain CL of the first antigen-binding moiety, the amino acid at position 124 is substituted with lysine (K) (Kabat numbering), the amino acid at position 123 is substituted with arginine (R) (Kabat numbering), and in the constant domain CH1 of the first antigen-binding moiety, the amino acid at position 147 is substituted with glutamic acid (E) (Kabat EU index numbering), and the amino acid at position 213 is substituted with glutamic acid (E) (Kabat EU index numbering).

[0116] In a particular embodiment, when the amino acid substitution according to the above embodiment is made in the constant domain CL and constant domain CH1 of the first antigen-binding moiety, the constant domain CL of the first antigen-binding moiety is a kappa isotype.

[0117] Alternatively, the amino acid substitutions according to the above embodiment may be made in the constant domains CL and CH1 of the second antigen-binding moiety instead of the constant domains CL and CH1 of the first antigen-binding moiety. In certain such embodiments, the constant domain CL of the second antigen-binding moiety is a kappa isotype.

[0118] Therefore, in one embodiment, in the constant domain CL of the second antigen-binding moiety, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering), and in the constant domain CH1 of the second antigen-binding moiety, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0119] In further embodiments, in the constant domain CL of the second antigen-binding moiety, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering), and in the constant domain CH1 of the second antigen-binding moiety, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0120] In yet another embodiment, in the constant domain CL of the second antigen-binding moiety, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering), the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering), in the constant domain CH1 of the second antigen-binding moiety, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0121] In one embodiment, in the constant domain CL of the second antigen-binding moiety, the amino acid at position 124 is substituted with lysine (K) (Kabat numbering), the amino acid at position 123 is substituted with lysine (K) (Kabat numbering), in the constant domain CH1 of the second antigen-binding moiety, the amino acid at position 147 is substituted with glutamic acid (E) (Kabat EU index numbering), and the amino acid at position 213 is substituted with glutamic acid (E) (Kabat EU index numbering).

[0122] In another embodiment, in the constant domain CL of the second antigen-binding moiety, the amino acid at position 124 is substituted with lysine (K) (Kabat numbering), the amino acid at position 123 is substituted with arginine (R) (Kabat numbering), and in the constant domain CH1 of the second antigen-binding moiety, the amino acid at position 147 is substituted with glutamic acid (E) (Kabat EU index numbering), and the amino acid at position 213 is substituted with glutamic acid (E) (Kabat EU index numbering).

[0123] To improve the heterodimerization of the Fc domain of these asymmetric (heterodimerized) proteins, in one embodiment according to these aspects of the present invention, in the first subunit of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), in the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V), optionally the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the Kabat EU index).

[0124] In further embodiments of these aspects of the present invention, in the first subunit of the Fc domain, the serine residue at position 354 is additionally replaced with a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 is additionally replaced with a cysteine ​​residue (E356C) (in particular, the serine residue at position 354 is additionally replaced with a cysteine ​​residue), and in the second subunit of the Fc domain, the tyrosine residue at position 349 is additionally replaced with a cysteine ​​residue (Y349C) (numbered according to the Kabat EU index).

[0125] Alternative heterodimerization techniques are described below under "Fc domain" and are intended as further embodiments of the present invention.

[0126] In further embodiments according to these aspects of the present invention, the Fc domain is a human IgG1Fc domain.

[0127] Fc domain and modification In certain embodiments, the bispecific antibody of the present invention includes an Fc domain composed of a first and a second subunit. It is understood that the features of the Fc domain described herein in relation to the bispecific antibody may be equally applicable to the Fc domain contained in the antibody of the present invention.

[0128] The Fc domain of a bispecific antibody consists of a pair of polypeptide chains containing the heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, and each subunit contains the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain can stably associate with each other. In one embodiment, the bispecific antibody of the present invention does not contain more than one Fc domain.

[0129] In one embodiment, the Fc domain of the bispecific antibody is an IgG Fc domain. In a particular embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain containing an amino acid substitution at position S228, in particular the amino acid substitution S228P (Kabat EU numbering). This amino acid substitution reduces Fab arm exchange of the IgG4 antibody in vivo (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further specific embodiment, the Fc domain is a human Fc domain. In an even more specific embodiment, the Fc domain is a human IgG1 Fc domain.

[0130] The Fc domains of IgG isotypes are characterized by various properties based on their interactions with, for example, Fc gamma receptors or neonatal Fc receptors (FcRn) (see, e.g., Vidarsson et al.; Front Immunol 5 (2014) Article 520, 1-17).

[0131] Fc domain modification that promotes heterodimerization The bispecific antibody according to the present invention comprises different antigen-binding moieties that can fuse to one or the other of two subunits of the Fc domain, and therefore the two subunits of the Fc domain are typically contained in two non-identical polypeptide chains. Co-recombinant expression of these polypeptides and subsequent dimerization result in several possible combinations of the two polypeptides. To improve the yield and purity of the bispecific antibody in recombinant production, it is advantageous to introduce modifications to the Fc domain of the bispecific antibody that promote the association of the desired polypeptide.

[0132] Therefore, in certain embodiments, the Fc domain of the bispecific antibody according to the present invention includes a modification that facilitates the association of the first and second subunits of the Fc domain. The site of the longest protein-protein interaction between the two subunits of the human IgG Fc domain is located within the CH3 domain of the Fc domain. Therefore, in one embodiment, the modification is located within the CH3 domain of the Fc domain.

[0133] To enhance heterodimerization, several methods exist for modifying the CH3 domain of the Fc domain, which are well described, for example, in International Publications 96 / 27011, 98 / 050431, European Patent No. 1870459, 2007 / 110205, 2007 / 147901, 2009 / 089004, 2010 / 129304, 2011 / 90754, 2011 / 143545, 2012058768, 2013157954, and 2013096291. Typically, in all such methods, the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are both manipulated in a complementary manner so that each CH3 domain (or the heavy chain containing it) does not homodimerize with itself but heterodimerizes with other complementaryly manipulated CH3 domains (resulting in heterodimerization of the first and second CH3 domains, and no homodimer is formed between the two first CH3 domains or the two second CH3 domains). These different methods for improved heavy chain heterodimerization are considered as alternatives in combination with heavy-light chain modifications in bispecific antibodies to reduce heavy / light chain mispairing and Bence-Jones type byproducts (e.g., exchange / substitution of VH and VL in one binding arm, and introduction of substitution of charged amino acids with opposite charges at the CH1 / CL interface).

[0134] In a specific embodiment, the modification that facilitates the association of the first and second subunits of the Fc domain is a so-called "knob-into-hole" modification, which includes a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain.

[0135] The knob-into-hole technique is described, for example, in U.S. Patents 5,731,168, 7,695,936, Ridgway et al., Prot Eng 9,617-621 (1996), and Carter, J Immunol Meth 248,7-15 (2001). Generally, this method involves introducing a projection ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, so that the projection can be positioned within the cavity to promote heterodimerization and inhibit homodimerization. The projection is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity of the same or similar size as the protrusion is formed at the interface of the second polypeptide by replacing a larger amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0136] Therefore, in certain embodiments, in the CH3 domain of the first subunit of the Fc domain of the bispecific antibody, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a repositionable protrusion within the CH3 domain of the first subunit within a cavity in the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit, within which the protrusion in the CH3 domain of the first subunit is repositionable.

[0137] Preferably, the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0138] Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0139] The protrusions and cavities can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0140] In a specific embodiment, in the first subunit of the Fc domain ("knob" subunit) (the CH3 domain), the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the second subunit of the Fc domain ("hole" subunit) (the CH3 domain), the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, the threonine residue at position 366 is further replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the Kabat EU index).

[0141] In further embodiments, in the first subunit of the Fc domain, the serine residue at position 354 is further replaced with a cysteine ​​residue (S354C), or the glutamic acid residue at position 356 is replaced with a cysteine ​​residue (E356C) (particularly the serine residue at position 354 being replaced with a cysteine ​​residue), and in the second subunit of the Fc domain, the tyrosine residue at position 349 is further replaced with a cysteine ​​residue (Y349C) (numbered according to the Kabat EU index). The introduction of these two cysteine ​​residues results in the formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0142] In certain embodiments, the first subunit of the Fc domain includes the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain includes the amino acid substitutions Y349C, T366S, L368A and Y407V (numbered according to the Kabat EU index).

[0143] In certain embodiments, the antigen-binding moiety that binds to the second antigen is fused to a first subunit of the Fc domain (including the "knob" modification) (optionally via the first antigen-binding moiety that binds to CCL2 and / or a peptide linker). While we do not wish to be bound by theory, the fusion of the antigen-binding moiety that binds to the second antigen (e.g., activated T cell antigen) to the knob-containing subunit of the Fc domain further minimizes the production of an antibody containing two antigen-binding moieties that bind to the activated T cell antigen (steric collision of the two knob-containing polypeptides).

[0144] Other techniques for CH3 modification that enhance heterodimerization have been considered as alternatives to the present invention and are described, for example, in International Publication Nos. 96 / 27011, 98 / 050431, European Patent No. 1870459, International Publication Nos. 2007 / 110205, 2007 / 147901, 2009 / 089004, 2010 / 129304, 2011 / 90754, 2011 / 143545, 2012 / 058768, 2013 / 157954, and 2013 / 096291.

[0145] In one embodiment, the heterodimerization method described in European Patent No. 1870459 is used instead. This method is based on the introduction of oppositely charged amino acids to specific amino acid positions at the CH3 / CH3 domain interface between two subunits of the Fc domain. A preferred embodiment of the bispecific antibody of the present invention is an amino acid mutation R409D, K370E in one of the two CH3 domains (of the Fc domain) and an amino acid mutation D399K, E357K in the other CH3 domain of the Fc domain (numbered according to the Kabat EU index).

[0146] In another embodiment, the bispecific antibodies of the present invention include amino acid mutations T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations T366S, L368A, and Y407V in the CH3 domain of the second subunit of the Fc domain, and additionally, amino acid mutations R409D and K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K and E357K in the CH3 domain of the second subunit of the Fc domain (numbered according to the Kabat EU index).

[0147] In another embodiment, the bispecific antibody of the present invention comprises amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or the bispecific antibody comprises amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and additionally comprises amino acid mutations R409D, K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K, E357K in the CH3 domain of the second subunit of the Fc domain (all numbered according to the Kabat EU index).

[0148] In one embodiment, the heterodimerization method described in International Publication No. 2013 / 157953 is used instead. In one embodiment, the first CH3 domain contains the amino acid mutation T366K, and the second CH3 domain contains the amino acid mutation L351D (numbered according to the Kabat EU index). In a further embodiment, the first CH3 domain contains a further amino acid mutation L351K. In a further embodiment, the second CH3 domain further contains an amino acid mutation selected from Y349E, Y349D, and L368E (preferably L368E) (numbered according to the Kabat EU index).

[0149] In one embodiment, the heterodimerization method described in International Publication No. 2012 / 058768 is used instead. In one embodiment, the first CH3 domain contains amino acid mutations L351Y, Y407A, and the second CH3 domain contains amino acid mutations T366A, K409F. In further embodiments, the second CH3 domain contains further amino acid mutations located at positions T411, D399, S400, F405, N390 or K392, e.g., (a) T411N, T411R, T411Q, T411K, T411D, T411E or T411W, (b) D399R, D399W, D399Y or (c) S400E, S400D, S400R or S400K, (d) F405I, F405M, F405T, F405S, F405V or F405W, (e) N390R, N390K or N390D, (f) K392V, K392M, K392R, K392L, K392F or K392E (numbered according to the Kabat EU index). In further embodiments, the first CH3 domain includes amino acid mutations L351Y and Y407A, and the second CH3 domain includes amino acid mutations T366V and K409F. In further embodiments, the first CH3 domain includes amino acid mutation Y407A, and the second CH3 domain includes amino acid mutations T366A and K409F. In further embodiments, the second CH3 domain further includes amino acid mutations K392E, T411E, D399R, and S400R (numbered according to the Kabat EU index).

[0150] In one embodiment, a heterodimerization method described in International Publication No. 2011 / 143545 is used instead, having, for example, amino acid modifications at positions selected from the group consisting of 368 and 409 (numbered according to the Kabat EU index).

[0151] In one embodiment, a heterodimerization technique described in International Publication No. 2011 / 090762 is used instead, which also employs the technique of inserting a knob into the hole described above. In one embodiment, the first CH3 domain contains the amino acid mutation T366W, and the second CH3 domain contains the amino acid mutation Y407A. In another embodiment, the first CH3 domain contains the amino acid mutation T366Y, and the second CH3 domain contains the amino acid mutation Y407T (numbered according to the Kabat EU index).

[0152] In one embodiment, the bispecific antibody or its Fc domain is of the IgG2 subclass, and the heterodimerization method described in International Publication No. 2010 / 129304 is used as an alternative.

[0153] In alternative embodiments, modifications that facilitate the association of the first and second subunits of the Fc domain include modifications that intervene in the electrostatic maneuvering effect, as described, for example, in PCT International Publication 2009 / 089004. Generally, this method involves the substitution of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues, such that homodimerization is electrostatically undesirable but heterodimerization is electrostatically desirable. In one such embodiment, the first CH3 domain includes an amino acid substitution with a negatively charged amino acid of K392 or N392 (e.g., glutamic acid (E) or aspartic acid (D), preferably K392D or N392D), and the second CH3 domain includes an amino acid substitution with a positively charged amino acid of D399, E356, D356 or E357 (e.g., lysine (K) or arginine (R), preferably D399K, E356K, D356K or E357K, more preferably D399K and E356K). In a further embodiment, the first CH3 domain further includes an amino acid substitution with a negatively charged amino acid of K409 or R409 (e.g., glutamic acid (E) or aspartic acid (D), preferably K409D or R409D). In further embodiments, the first CH3 domain further comprises, or is otherwise, an amino acid substitution with negatively charged amino acids at K439 and / or K370 (e.g., glutamic acid (E) or aspartic acid (D)) (all numbered according to the Kabat EU index).

[0154] In further embodiments, the heterodimerization method described in International Publication No. 2007 / 147901 is used instead. In one embodiment, the first CH3 domain contains amino acid mutations K253E, D282K, and K322D, and the second CH3 domain contains amino acid mutations D239K, E240K, and K292D (numbered according to the Kabat EU index).

[0155] In yet another embodiment, the heterodimerization method described in International Publication No. 2007 / 110205 may be used instead.

[0156] In one embodiment, the first subunit of the Fc domain includes amino acid substitutions K392D and K409D, and the second subunit of the Fc domain includes amino acid substitutions D356K and D399K (numbered according to the Kabat EU index).

[0157] As used herein with respect to bispecific anti-CCL2 antibodies, the term “wild-type (WT) IgG or IgG1” refers to a bispecific antibody comprising an IgG or IgG1 heavy chain that may include the modifications / mutations described above that promote heterodimerization, but does not contain further Fc domain modifications / mutations that increase or decrease Fc receptor binding and / or effector function as described below.

[0158] Fc domain modifications / mutations that increase or decrease Fc receptor binding and / or effector function: Modification of bispecific anti-CCL2 antibodies by sweeping technology By modifying a bispecific anti-CCL2 antibody using a sweeping technique, we enabled the bispecific anti-CCL2 antibody to eliminate free CCl2 over a long period, thereby sustaining biological effects such as anticancer efficacy in vivo.

[0159] The concept of sweeping is described, for example, in Igawa et al, Immunological Reviews 270 (2016) 132-151, WO2012 / 122011, WO2016 / 098357, and WO2013 / 081143, which are incorporated herein by reference.

[0160] The present invention provides a method for promoting antibody-mediated antigen uptake into cells by reducing the antigen-binding activity (binding ability) of the antibody in the acidic pH range compared to its antigen-binding activity in the neutral pH range. This promotes antigen uptake into cells. The present invention also provides a method for promoting antibody-mediated antigen uptake into cells, based on altering at least one amino acid in the antigen-binding domain of the antibody that promotes antigen uptake into cells. The present invention also provides a method for promoting antibody-mediated antigen uptake into cells, based on substituting at least one amino acid with histidine, or inserting at least one histidine into the antigen-binding domain of the antibody that promotes antigen uptake into cells.

[0161] In this specification, "antigen uptake into cells" mediated by antibodies means that antigens are taken up into cells by endocytosis. Furthermore, in this specification, "promoting uptake into cells" means that the rate of intracellular uptake of antibodies bound to antigens in plasma is increased, and / or the amount of recycled uptaken antigens into plasma is reduced. This means that the rate of uptake into cells is promoted compared to antibodies before increasing their human FcRn binding activity in the neutral pH range, or before increasing their human FcRn binding activity and reducing their antigen-binding activity (binding ability) in the acidic pH range to less than their antigen-binding activity in the neutral pH range. This rate is preferably improved compared to intact human IgG, and more preferably compared to intact human IgG. Therefore, in this invention, whether antigen uptake into cells is promoted by antibodies can be evaluated based on the increase in the rate of antigen uptake into cells. The rate of antigen uptake into cells can be calculated, for example, by monitoring the decrease in antigen concentration in the culture medium over time after adding the antigen and antibody to a culture medium containing human FcRn-expressing cells, or by monitoring the amount of antigen uptake by human FcRn-expressing cells over time. Using the method of the present invention to promote antibody-mediated antigen uptake into cells, for example, the rate of antigen removal from plasma can be increased by administering an antibody. Therefore, whether antibody-mediated antigen uptake into cells is promoted can also be evaluated, for example, by testing whether the rate of antigen removal from plasma is accelerated, or whether the total antigen concentration in plasma decreases by administering an antibody.

[0162] Here, "total antigen concentration in plasma" refers to the sum of antibody-bound antigen and unbound antigen concentrations, or "free antigen concentration in plasma," which is the concentration of unbound antigen. Various methods for measuring "total antigen concentration in plasma" or "free antigen concentration in plasma" are well known in the art, as described below.

[0163] As used herein, “intact human IgG” (or “wild-type (WT) human IgG” means unmodified human IgG (except for the potential modifications of heterodimerization described above) and is not limited to any particular class of IgG. This means that human IgG1, IgG2, IgG3, or IgG4 can be used as “intact human IgG” as long as they can bind to human FcRn in an acidic pH range. Preferably, “intact human IgG” may be human IgG1.

[0164] The present invention also provides methods for increasing the number of antigens to which a single antibody can bind. More specifically, the present invention provides a method for increasing the number of antigens to which a single antibody having human FcRn binding activity in an acidic pH range can bind by increasing the human FcRn binding activity of the antibody in a neutral pH range. The present invention also provides a method for increasing the number of antigens to which a single antibody having human FcRn binding activity in an acidic pH range can bind by altering at least one amino acid in the human FcRn binding domain of the antibody.

[0165] The present invention provides a method for promoting antibody-mediated antigen uptake into cells. More specifically, the present invention provides a method for promoting antigen uptake into cells by an antibody having human FcRn binding activity in an acidic pH range, which is based on increasing the human FcRn binding activity of the antibody in a neutral pH range. The present invention also provides a method for improving antigen uptake into cells by an antibody having human FcRn binding activity in an acidic pH range, which is based on altering at least one amino acid in the human FcRn binding domain of the antibody.

[0166] The present invention also provides a method for promoting antigen uptake into cells by an antibody having human FcRn binding activity in an acidic pH range, wherein the parent IgG has a human FcRn binding domain that includes the Fc domain of the parent IgG. The present invention provides a method based on using a human FcRn-binding domain that contains an amino acid sequence in which at least one amino acid selected from positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434 and 436 (EU numbering) is substituted with a different amino acid.

[0167] The present invention also provides a method for promoting antibody-mediated antigen uptake into cells by reducing the antigen-binding activity (binding ability) of the antibody in the acidic pH range compared to its antigen-binding activity in the neutral pH range. This promotes antigen uptake into cells. The present invention also provides a method for promoting antibody-mediated antigen uptake into cells, based on altering at least one amino acid in the antigen-binding domain of the antibody that promotes antigen uptake into cells. The present invention also provides a method for promoting antibody-mediated antigen uptake into cells, based on substituting at least one amino acid with histidine, or inserting at least one histidine into the antigen-binding domain of the antibody that promotes antigen uptake into cells.

[0168] In this specification, "antigen uptake into cells" mediated by antibodies means that antigens are taken up into cells by endocytosis. Furthermore, in this specification, "promoting uptake into cells" means that the rate of intracellular uptake of antibodies bound to antigens in plasma is increased, and / or the amount of recycled uptaken antigens into plasma is reduced. This means that the rate of uptake into cells is promoted compared to antibodies before increasing their human FcRn binding activity in the neutral pH range, or before increasing their human FcRn binding activity and reducing their antigen-binding activity (binding ability) in the acidic pH range to less than their antigen-binding activity in the neutral pH range. This rate is preferably improved compared to intact human IgG, and more preferably compared to intact human IgG. Therefore, in this invention, whether antigen uptake into cells is promoted by antibodies can be evaluated based on the increase in the rate of antigen uptake into cells. The rate of antigen uptake into cells can be calculated, for example, by monitoring the decrease in antigen concentration in the culture medium over time after adding the antigen and antibody to a culture medium containing human FcRn-expressing cells, or by monitoring the amount of antigen uptake by human FcRn-expressing cells over time. Using the method of the present invention to promote antibody-mediated antigen uptake into cells, for example, the rate of antigen removal from plasma can be increased by administering an antibody. Therefore, whether antibody-mediated antigen uptake into cells is promoted can also be evaluated, for example, by testing whether the rate of antigen removal from plasma is accelerated, or whether the total antigen concentration in plasma decreases by administering an antibody.

[0169] Here, "total antigen concentration in plasma" refers to the sum of antibody-bound antigen and unbound antigen concentrations, or "free antigen concentration in plasma," which is the concentration of unbound antigen. Various methods for measuring "total antigen concentration in plasma" or "free antigen concentration in plasma" are well known in the art, as described below.

[0170] As used herein, “intact human IgG” (or “wild-type IgG”) means unmodified human IgG (excluding the potential modifications of heterodimerization described above) and is not limited to any particular class of IgG. This means that human IgG1, IgG2, IgG3, or IgG4 can be used as “intact human IgG” as long as they can bind to human FcRn in an acidic pH range. Preferably, “intact human IgG” may be human IgG1.

[0171] As used herein, “parent IgG” means unmodified IgG that is later modified to produce a variant, insofar as the modified variant of parent IgG can bind to human FcRn in an acidic pH range (and therefore parent IgG does not necessarily require binding activity to human FcRn under acidic conditions). Parent IgG may be naturally occurring IgG, or a variant or modified version of naturally occurring IgG. Parent IgG may refer to the polypeptide itself, a composition containing parent IgG, or the amino acid sequence encoding it. It should be noted that “parent IgG” includes known commercially available recombinant-producing IgG, as outlined below. The origin of “parent IgG” is not limited and can be obtained from any non-human animal or human organism. Preferably, the organism is selected from mice, rats, guinea pigs, hamsters, gerbils, cats, rabbits, dogs, goats, sheep, cattle, horses, camels, and non-human primates. In another embodiment, “parent IgG” can also be obtained from cynomolgus macaques, marmosets, rhesus macaques, chimpanzees, or humans. Preferably, the “parent IgG” is obtained from human IgG1, but is not limited to a specific class of IgG. This means that human IgG1, IgG2, IgG3, or IgG4 can be used as “parent IgG” as appropriate. Similarly, any class or subclass of IgG from any of the above-mentioned biological sources can preferably be used as “parent IgG.” Examples of naturally occurring variants or manipulated versions of IgG are described, but are not limited to, Curr Opin Biotechnol. 2009 Dec;20(6):685-91, Curr Opin Immunol. 2008 Aug;20(4):460-70, Protein Eng Des Sel. 2010 Apr;23(4):195-202, WO 2009 / 086320, WO 2008 / 092117, WO 2007 / 041635, and WO 2006 / 105338.

[0172] The present invention also provides a method for increasing the ability of an antibody to remove plasma antigens by administration. In the present invention, “a method for increasing the ability to remove plasma antigens” is synonymous with “a method for enhancing the ability of an antibody to remove antigens from plasma.” More specifically, the present invention provides a method for increasing the ability of an antibody having human FcRn binding activity in an acidic pH range to remove plasma antigens by increasing the human FcRn binding activity of the antibody in a neutral pH range. The present invention also provides a method for increasing the ability of an antibody having human FcRn binding activity in an acidic pH range to remove plasma antigens, the method being based on altering at least one amino acid in the human FcRn binding domain of the antibody.

[0173] This invention also relates to the parent IgG of a human FcRn-binding domain containing the parent IgG Fc domain. This invention provides a method for increasing the ability of an antibody having human FcRn-binding activity to remove plasma antigens in an acidic pH range by using a human FcRn-binding domain containing an amino acid sequence in which at least one amino acid selected from positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (EU numbering) in the Fc domain is substituted with a different amino acid.

[0174] The present invention also provides a method for increasing the ability of an antibody to eliminate plasma antigens by reducing the antigen-binding activity in the acidic pH range of the antibody, which has improved ability to eliminate plasma antigens compared to its antigen-binding activity in the neutral pH range. The present invention also provides a method for increasing the ability of an antibody to eliminate plasma antigens by altering at least one amino acid in the antigen-binding domain of the antibody, which has improved ability to eliminate plasma antigens. The present invention also provides a method for increasing the ability to eliminate plasma antigens by administering the antibody, by substituting at least one amino acid with histidine, or by inserting at least one histidine into the antigen-binding domain of the antibody, which has improved ability to eliminate plasma antigens.

[0175] In this specification, "ability to remove plasma antigens" means the ability to remove antigens from plasma when an antibody is administered or secreted in vivo. Therefore, "increase in the ability of an antibody to remove plasma antigens" in this specification means that the rate of antigen removal from plasma is accelerated when the antibody is administered, compared to before increasing the human FcRn binding activity of the antibody in the neutral pH range, or before increasing the human FcRn binding activity while simultaneously decreasing its antigen-binding activity in the acidic pH range compared to that in the neutral pH range. The increase in the activity of an antibody for removing antigens from plasma can be evaluated, for example, by administering a soluble antigen and antibody in vivo and measuring the concentration of the soluble antigen in the plasma after administration. If the concentration of the soluble antigen in the plasma after administration of the soluble antigen and antibody decreases by increasing the human FcRn binding activity of the antibody in the neutral pH range, or by increasing its human FcRn binding activity while simultaneously decreasing its antigen-binding activity in the acidic pH range compared to that in the neutral pH range, then the ability of the antibody to remove plasma antigens can be determined to have increased. Soluble antigens can take the form of antibody-bound antigens or non-binding antigens, and their concentrations can be determined as "antibody-bound antigen concentration in plasma" and "non-binding antigen concentration in plasma," respectively (the latter being synonymous with "free antigen concentration in plasma"). Since "total antigen concentration in plasma" means the sum of antibody-bound and non-binding antigen concentrations, or the "free antigen concentration in plasma" which is the non-binding antigen concentration, the concentration of soluble antigens can be determined as the "total antigen concentration in plasma." Various methods for measuring "total antigen concentration in plasma" or "free antigen concentration in plasma" are well known in the art, as described below.

[0176] The present invention also provides methods for improving the pharmacokinetics of antibodies. More specifically, the present invention provides a method for improving the pharmacokinetics of antibodies having human FcRn binding activity in an acidic pH range by increasing the human FcRn binding activity of the antibody in a neutral pH range. Furthermore, the present invention provides a method for improving the pharmacokinetics of antibodies having human FcRn binding activity in an acidic pH range by altering at least one amino acid in the human FcRn binding domain of the antibody.

[0177] This invention also relates to the parent IgG of a human FcRn-binding domain containing the Fc domain of IgG. This invention provides a method for improving the pharmacokinetics of an antibody having human FcRn-binding activity in an acidic pH range by using a human FcRn-binding domain containing an amino acid sequence in which at least one amino acid selected from positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (EU numbering) is substituted with a different amino acid.

[0178] The ratio of the free antigen concentration to the plasma concentration or total concentration of free antigen not bound to an antibody can be determined by methods known to those skilled in the art, for example, by the method described in Pharm Res. 2006 Jan;23(1):95-103. Alternatively, if the antigen exhibits a specific function in vivo, it can be evaluated whether the antigen is bound to an antibody (antagonist molecule) that neutralizes the antigen function by testing whether the antigen function is neutralized. Whether the antigen function is neutralized can be evaluated by assaying an in vivo marker that reflects the antigen function. Whether the antigen is bound to an antibody (agonist molecule) that activates the antigen function can be evaluated by assaying an in vivo marker that reflects the antigen function.

[0179] Determination of the plasma concentration of free antigen and the ratio of the amount of free antigen in plasma to the amount of total antigen in plasma, in vivo marker assays, and such measurements are not particularly limited, but are preferably performed after a certain period of time has elapsed since antibody administration. In the present invention, the period after antibody administration is not particularly limited. Those skilled in the art can determine an appropriate period depending on the properties of the antibody to be administered. Such periods include, for example, 1 day after antibody administration, 3 days after antibody administration, 7 days after antibody administration, 14 days after antibody administration, and 28 days after antibody administration. Here, "plasma antigen concentration" means either "total antigen concentration in plasma," which is the sum of antibody-bound antigen and unbound antigen concentrations, or "free antigen concentration in plasma," which is the antibody-unbound antigen concentration.

[0180] The total antigen concentration in plasma can be reduced by 2, 5, 10, 20, 50, 100, 200, 500, 1,000 times, or more, by administration of the antibody of the present invention compared to administration of a reference antibody containing an intact human IgG Fc domain as a human FcRn binding domain, or compared to a case where the antigen-binding domain molecule of the present invention is not administered.

[0181] In another embodiment, the present invention provides a bispecific anti-CCL2 antibody exhibiting pH-dependent binding properties. As used herein, the expression “pH-dependent binding” means that the antibody exhibits “reduced binding to CCL2 at acidic pH compared to binding at neutral pH” (both expressions may be used interchangeably for the purposes of this disclosure). For example, an antibody “having pH-dependent binding properties” includes an antibody that binds to CCL2 with higher affinity at neutral pH than at acidic pH. In certain embodiments, the bispecific antibody of the present invention binds to CCL2 with at least 2x, 3x, 5x, 10x, 15x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 55x, 60x, 65x, 70x, 75x, 80x, 85x, 90x, 95x, 100x, 200x, 400x, 1000x, 10000x or more affinity at neutral pH than at acidic pH. In some embodiments, the antibody binds to CCL2 with higher affinity at pH 7.4 than at pH 5.8. In further embodiments, the antibody binds to CCL2 with at least 2x, 3x, 5x, 10x, 15x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 55x, 60x, 65x, 70x, 75x, 80x, 85x, 90x, 95x, 100x, 200x, 400x, 1000x, 10000x or more affinity at pH 7.4 than at pH 5.8.

[0182] If the antigen is a soluble protein, the antibody can have a longer plasma half-life than the antigen itself and can function as a carrier of the antigen. Therefore, antibody binding to the antigen can result in an extension of the antigen's half-life in plasma (i.e., a decrease in the clearance of the antigen from the plasma). This is due to the reuse of the antigen-antibody complex by FcRn via the intracellular endosomal pathway (Roopenian, Nat. Rev. Immunol. 7(9):715-725 (2007)). However, antibodies with pH-dependent binding properties, which bind to the antigen in a neutral extracellular environment and release the antigen into the acidic endosomal compartment after entering the cell, are expected to have superior antigen neutralization and clearance properties compared to their pH-independent binding counterparts (Igawa et al., Nature Biotechnol. 28(11):1203-1207(2010); Devanaboyina et al., mAbs 5(6):851-859(2013); WO 2009 / 125825).

[0183] For the purposes of this disclosure, the “affinity” of an antibody for CCL2 is expressed in terms of the antibody’s KD. The KD of an antibody refers to the equilibrium dissociation constant of the antibody-antigen interaction. The higher the KD value of an antibody that binds to an antigen, the weaker its binding affinity is to that particular antigen. Therefore, as used herein, the expression “higher affinity at neutral pH than at acidic pH” (or the equivalent expression “pH-dependent binding”) means that the KD of an antibody that binds to CCL2 at acidic pH is greater than the KD of an antibody that binds to CCL2 at neutral pH. For example, in the context of the present invention, if the KD of an antibody that binds to CCL2 at acidic pH is at least twice as high as the KD of an antibody that binds to CCL2 at neutral pH, then the antibody is considered to bind to CCL2 with higher affinity at neutral pH than at acidic pH. Accordingly, the present invention includes an antibody that binds to CCL2 at an acidic pH with a KD of at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more than the KD of the antibody that binds to CCL2 at a neutral pH. In another embodiment, the KD value of the antibody at a neutral pH may be 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, 10⁻¹² M or less. In another embodiment, the KD value of the antibody at an acidic pH may be 10⁻⁹ M, 10⁸ M, 10⁷ M, 10⁻⁶ M or more.

[0184] In further embodiments, if the KD of an antibody that binds to CCL2 at pH 5.8 is at least twice as large as the KD of an antibody that binds to CCL2 at pH 7.4, the antibody is considered to bind with higher affinity at neutral pH than at acidic pH. In some embodiments, the provided antibody binds to CCL2 at pH 5.8 with a KD at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more than the KD of an antibody that binds to CCL2 at pH 7.4. In another embodiment, the KD values ​​of the antibody at pH 7.4 may be 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, 10⁻¹² M or less. In yet another embodiment, the KD values ​​of the antibody at pH 5.8 may be 10⁻⁹ M, 10⁸ M, 10⁷ M, 10⁻⁶ M or more.

[0185] The binding characteristics of an antibody to a particular antigen can also be expressed in terms of the antibody's kd. The antibody's kd refers to the dissociation rate constant of the antibody to a particular antigen and is expressed as the reciprocal of seconds (i.e., seconds - 1). An increase in the kd value means that the antibody binds to that antigen more weakly. Therefore, the present invention includes antibodies that bind to CCL2 at acidic pH with a higher kd value than neutral pH. The present invention also includes antibodies that bind to CCL2 at acidic pH with a kd at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more than the kd of the antibody that binds to CCL2 at neutral pH. In another embodiment, the kd values ​​of the antibody at neutral pH may be 10⁻² ¹ / s, 10⁻³ ¹ / s, 10⁻⁴ ¹ / s, 10⁻⁵ ¹ / s, or 10⁻⁶ ¹ / s or less. In another embodiment, the kd values ​​of the antibody at acidic pH may be 10⁻³ ¹ / s, 10⁻² ¹ / s, or 10⁻¹ ¹ / s or more. The present invention also includes antibodies that bind to CCL2 with a higher kd value at pH 5.8 than at pH 7.4. The present invention includes an antibody that binds to CCL2 at pH 5.8 with a kd of at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more than the kd of the antibody that binds to CCL2 at pH 7.4. In another embodiment, the kd value of the antibody at pH 7.4 may be 10⁻² ¹ / s, 10⁻³ ¹ / s, 10⁻⁴ ¹ / s, 10⁻⁵ ¹ / s, or 10⁻⁶ ¹ / s or less. In another embodiment, the kd value of the antibody at pH 5.8 may be 10⁻³ ¹ / s, 10⁻² ¹ / s, or 10⁻¹ ¹ / s or more.

[0186] In certain cases, "decreased binding to CCL2 at acidic pH compared to binding at neutral pH" is expressed in terms of the ratio of the KD value of the antibody binding to CCL2 at acidic pH to the KD value of the antibody binding to CCL2 at neutral pH (or vice versa). For example, if an antibody exhibits an acidic / neutral KD ratio of 2 or higher, for the purposes of the present invention, the antibody may be considered to exhibit "decreased binding to CCL2 at acidic pH compared to binding at neutral pH." In certain embodiments, the KD ratio at pH 5.8 / pH 7.4 for the anti-CCL2 antibody of the present invention is 2 or higher. In certain exemplary embodiments, the acid / neutral KD ratio for the antibody of the present invention may be 2x, 3x, 5x, 10x, 15x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 55x, 60x, 65x, 70x, 75x, 80x, 85x, 90x, 95x, 100x, 200x, 400x, 1000x, 10000x or more. In another embodiment, the KD value of the antibody at neutral pH may be 10⁻⁷M, 10⁻⁸M, 10⁻⁹M, 10⁻¹⁰M, 10⁻¹¹M, 10⁻¹²M or less. In another embodiment, the KD value of the antibody at acidic pH may be 10⁻⁹M, 10⁸M, 10⁷M, 10⁻⁶M or more. In a further example, if an antibody exhibits a KD ratio of 2 or greater at pH 5.8 / pH 7.4, the antibody may be considered to exhibit "decreased binding to CCL2 at acidic pH compared to binding at neutral pH." In certain exemplary embodiments, the KD ratio of an antibody at pH 5.8 / pH 7.4 may be 3x, 5x, 10x, 15x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 55x, 60x, 65x, 70x, 75x, 80x, 85x, 90x, 95x, 100x, 200x, 400x, 1000x, 10000x or greater. In another embodiment, the KD value of an antibody at pH 7.4 may be 10⁻⁷M, 10⁻⁸M, 10⁻⁹M, 10⁻¹M, 10⁻¹²M or less. In another embodiment, the KD value of the antibody at pH 5.8 may be 10⁻⁹ M, 10⁻⁸ M, 10⁷ M, 10⁶ M or higher.

[0187] In certain cases, "decreased binding to CCL2 at acidic pH compared to binding at neutral pH" is expressed in terms of the ratio of the kd value of the antibody binding to CCL2 at acidic pH to the kd value of the antibody binding to CCL2 at neutral pH (or vice versa). For example, if an antibody exhibits an acidic / neutral kd ratio of 2 or greater, for the purposes of the present invention, the antibody may be considered to exhibit "decreased binding to CCL2 at acidic pH compared to binding at neutral pH." In certain exemplary embodiments, the pH 5.8 / pH 7.4 kd ratio of the antibody of the present invention is 2 or greater. In certain exemplary embodiments, the acid / neutral kd ratio for the antibody of the present invention may be 2x, 3x, 5x, 10x, 15x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 55x, 60x, 65x, 70x, 75x, 80x, 85x, 90x, 95x, 100x, 200x, 400x, 1000x, 10000x or more. In another embodiment, the kd value of the antibody at neutral pH may be 10⁻² ¹ / s, 10⁻³ ¹ / s, 10⁻⁴ ¹ / s, 10⁻⁵ ¹ / s, 10⁻⁶ ¹ / s or less. In another embodiment, the kd value of the antibody at acidic pH may be 10⁻³ ¹ / s, 10⁻² ¹ / s, 10⁻¹ ¹ / s or more. In certain exemplary embodiments, the pH 5.8 / pH 7.4 kd ratio of the antibody of the present invention may be 2x, 3x, 5x, 10x, 15x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 55x, 60x, 65x, 70x, 75x, 80x, 85x, 90x, 95x, 100x, 200x, 400x, 1000x, 10000x or more. In another embodiment, the kd value of the antibody at pH 7.4 may be 10⁻² ¹ / s, 10⁻³ ¹ / s, 10⁻⁴ ¹ / s, 10⁻⁵ ¹ / s, 10⁻⁶ ¹ / s or less. In another embodiment, the kd value of the antibody at pH 5.8 may be 10⁻³ ¹ / s, 10⁻² ¹ / s, 10⁻¹ ¹ / s or more.

[0188] As used herein, the term "acidic pH" means a pH of 4.0 to 6.5. The term "acidic pH" includes any one of the following pH values: 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5. In certain embodiments, "acidic pH" is 5.8.

[0189] As used herein, the term "neutral pH" means a pH of 6.7 to about 10.0. The term "neutral pH" includes any one of the following pH values: 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and 10.0. In certain embodiments, "neutral pH" is 7.4.

[0190] The KD and kd values ​​expressed herein can be determined using a surface plasmon resonance-based biosensor to characterize antibody-antigen interactions. The KD and kd values ​​can be determined at 25°C or 37°C.

[0191] In a further embodiment, the present invention provides bispecific anti-CCL2 antibodies that form an immune complex (i.e., an antigen-antibody complex) with CCL2. In certain embodiments, two or more bispecific anti-CCL2 antibodies bind to two or more CCL2 molecules to form an immune complex. This is possible because the antibody has two antigen-binding sites, while CCL2 exists as a homodimer containing two CCL2 molecules.

[0192] Generally speaking, when two or more antibodies form an immune complex with two or more antigens, the resulting immune complex can strongly bind to Fc receptors present on the cell surface via the avidity effect mediated by the Fc domain of the antibodies in the complex, and can then be efficiently taken up into the cell. Therefore, the above-mentioned anti-CCL2 antibodies, which can form an immune complex containing two or more anti-CCL2 antibodies and two or more CCL2 molecules, can lead to rapid clearance of CCL2 from plasma in vivo via strong binding to Fc receptors through the avidity effect.

[0193] Furthermore, antibodies with pH-dependent binding properties are thought to have superior antigen neutralization and clearance properties compared to their pH-independent binding counterparts (Igawa et al., Nature Biotech. 28(11):1203-1207(2010); Devanaboyina et al. mAbs 5(6):851-859(2013); WO 2009 / 125825). Therefore, antibodies possessing both of the above properties, i.e., antibodies with pH-dependent binding properties that form immune complexes containing two or more antigens and two or more antibodies, are expected to have superior properties, such as highly promoting the removal of antigens from plasma (WO 2013 / 081143).

[0194] In one embodiment, the present invention provides a polypeptide comprising a plurality of variant Fc regions with enhanced Fc-gamma-RIIb binding activity, comprising at least two amino acid changes, including: (a) a change in one amino acid at position 236; and (b) a change in at least one amino acid at at least one position selected from the group consisting of: 231, 232, 233, 234, 235, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334, and 396 in EU numbering.

[0195] In one embodiment, the present invention provides a polypeptide comprising a variant Fc region with enhanced Fc-gamma-RIIb binding activity, which includes an amino acid change at position 236 in EU numbering.

[0196] In one embodiment, the present invention provides a polypeptide comprising a variant Fc region with enhanced Fc-gamma-RIIb binding activity, comprising at least two amino acid changes including: (a) a change in one amino acid at position 236, and (b) a change in at least one amino acid at at least one position selected from the group consisting of: 231, 232, 235, 239, 268, 295, 298, 326, 330, and 396 in EU numbering. In a further embodiment, the variant Fc region comprises an amino acid change at at least one position selected from the group consisting of: 231, 232, 235, 239, 268, 295, 298, 326, 330, and 396 in EU numbering. In a further embodiment, the variant Fc region comprises an amino acid change at at least one position selected from the group consisting of: 268, 295, 326, and 330 in EU numbering.

[0197] In another embodiment, the present invention provides a polypeptide comprising a variant Fc region having enhanced Fc-gamma-RIIb binding activity, comprising any one of the following amino acid changes (1) to (37). In EU numbering, (1) positions 231, 236, 239, 268 and 330; (2) positions 231, 236, 239, 268, 295 and 330; (3) positions 231, 236, 268 and 330; (4) positions 231, 236, 268, 295 and 330; (5) positions 232, 236, 239, 268, 295 and 330; (6) positions 232, 236, 268, 295 and 330; (7) positions 232, 236, 268 and 330; (8) positions 235, 236, 268, 295, 326 and 330; (9) position 2 35, 236, 268, 295 and 330; (10) Positions 235, 236, 268 and 330; (11) Positions 235, 236, 268, 330 and 396; (12) Positions 235, 236, 268 and 396; (13) Positions 236, 239, 268, 295, 298 and 330; (14) Positions 236, 239, 268, 295, 326 and 330; (15) Positions 236, 239, 268, 295 and 330; (16) Positions 236, 239, 268, 298 and 330; (17) Positions 236, 239, 268, 32 6 and 330; (18) Positions 236, 239, 268 and 330; (19) Positions 236, 239, 268, 330 and 396; (20) Positions 236, 239, 268 and 396; (21) Positions 236 and 268; (22) Positions 236, 268 and 295; (23) Positions 236, 268, 295, 298 and 330; (24) Positions 236, 268, 295, 326 and 330; (25) Positions 236, 268, 295, 326, 330 and 396; (26) Positions 236, 268, 295 and 330; (27) Position 236, 268, 295, 330 and 396; (28) positions 236, 268, 298 and 330; (29) positions 236, 268, 298 and 396; (30) positions 236, 268, 326 and 330; (31) positions 236, 268, 326, 330 and 396; (32) positions 236, 268 and 330; (33) positions 236, 268, 330 and 396; (34) positions 236, 268 and 396; (35) positions 236 and 295; (36) positions 236, 330 and 396; and (37) positions 236 and 396.

[0198] In further embodiments, the variant Fc region with enhanced Fc-gamma-RIIb binding activity comprises at least one amino acid selected from the group consisting of: (a) Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr (position 231); (b) Ala, Asp, Glu, Phe, Gly, His , Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr (232nd position); (c) Asp (233rd position); (d) Trp, Tyr (234th position); (e) Trp (23 5th place); (f) Ala, Asp, Glu, His, Ile, Leu, Met, Asn, Gln, Ser, Thr, Val (236th place); (g) Asp, Tyr (237th place); (h) Glu, Ile, Met, G ln, Tyr (238th place); (i) Ile, Leu, Asn, Pro, Val (239th place); (j) Ile (264th place); (k) Phe (266th place); (l) Ala, His, Leu (267th place); (m) As p, Glu (268th place); (n) Asp, Glu, Gly (271st place); (o) Leu (295th place); (p) Leu (298th place); (q) Glu, Phe, Ile, Leu (325th place); (r) Thr (326 position);(s)Ile, Asn(327th position);(t)Thr(328th position);(u)Lys, Arg(330th position);(v)Glu(331st position);(w)Asp(332nd position);(x)Asp, Ile, Met, Val , Tyr (position 334); and (y)Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr (position 396).

[0199] In further embodiments, the variant Fc region with enhanced Fc-gamma-RIIb binding activity comprises at least one amino acid change (e.g., substitution) selected from the group consisting of: (a) Gly, Thr (position 231); (b) Asp (position 232); (c) Trp (position 235); (d) Asn, Thr (position 236); (e) Val (position 239); (f) Asp, Glu (position 268); (g) Leu (position 295); (h) Leu (position 298); (i) Thr (position 326); (j) Lys, Arg (position 330); and (k) Lys, Met (position 396). In further embodiments, the variant Fc region with enhanced Fc-gamma-RIIb binding activity includes the following amino acid changes (e.g., substitutions): Asn at position 236, Glu at position 268, Lys at position 330, and Met at position 396, according to EU numbering. In further embodiments, the variant Fc region with enhanced Fc-gamma-RIIb binding activity includes the following amino acid changes (e.g., substitutions): Asn at position 236, Asp at position 268, and Lys at position 330, according to EU numbering. In further embodiments, the variant Fc region with enhanced Fc-gamma-RIIb binding activity includes the following amino acid changes (e.g., substitutions): Asn at position 236, Asp at position 268, Leu at position 295, and Lys at position 330, according to EU numbering. In further embodiments, the variant Fc region with enhanced Fc-gamma-RIIb binding activity includes the following amino acid changes (e.g., substitutions): Thr at position 236, Asp at position 268, and Lys at position 330, according to EU numbering. In further embodiments, the variant Fc region with enhanced Fc-gamma-RIIb binding activity includes the following amino acid changes (e.g., substitutions): Asn at position 236, Asp at position 268, Leu at position 295, Thr at position 326, and Lys at position 330, according to EU numbering. In further embodiments, the variant Fc region with enhanced Fc-gamma-RIIb binding activity includes the following amino acid changes (e.g., substitutions): Trp at position 235, Asn at position 236, Asp at position 268, Leu at position 295, Thr at position 326, and Lys at position 330, according to EU numbering.

[0200] In another embodiment, the present invention provides isolated polypeptides comprising a variant Fc region with an increased isoelectric point (pI). In certain embodiments, the variant Fc region described herein comprises at least two amino acid changes in the parent Fc region. In certain embodiments, each of the amino acid changes increases the isoelectric point (pI) of the variant Fc region compared to the parent Fc region. These are based on the finding that, for example, when an antibody is administered in vivo, an antibody with an increased pI due to modification of at least two amino acid residues can enhance antigen removal from plasma.

[0201] In this invention, pI may be a theoretically or experimentally determined pI. The value of pI can be determined, for example, by isoelectric focusing, which is known to those skilled in the art. The theoretical value of pI can be calculated, for example, using gene and amino acid sequence analysis software (such as Genetyx).

[0202] In one embodiment, the pI value may increase compared to before modification by, for example, at least 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 or more, at least 0.6, 0.7, 0.8, 0.9 or more, at least 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 or more, or at least 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0 or more.

[0203] In certain embodiments, amino acids for increasing pI can be exposed on the surface of the variant Fc region. In this invention, surface-exposed amino acids typically refer to amino acid residues located on the surface of the polypeptide constituting the variant Fc region. Surface-located amino acid residues refer to amino acid residues whose side chains can come into contact with solvent molecules (generally mostly water molecules). However, the side chains do not necessarily need to be in complete contact with the solvent molecules; if even a part of the side chain is in contact with the solvent molecules, the amino acid is defined as a "surface-located amino acid residue." Surface-located amino acid residues of polypeptides also include amino acid residues located near the surface, which may be affected by the charge from other amino acid residues whose side chains are in contact with solvent molecules, even if only partially. Those skilled in the art can prepare homology models of polypeptides using, for example, commercially available software. Alternatively, methods known to those skilled in the art, such as X-ray crystallography, can be used. Surface-exposed amino acid residues are determined using coordinates from a 3D model using, for example, a computer program such as the Insight II program (Accelrys). The surface-exposureable regions can be determined using algorithms known in the art (e.g., Lee and Richards (J.Mol.Biol.55:379-400(1971)); Connolly (J.Appl.Cryst.16:548-558(1983)). The surface-exposure regions can be determined using software suitable for protein modeling and three-dimensional structural information. Software available for such purposes includes, for example, the SYBYL Biopolymer Module software (Tripos Associates). If the algorithm requires a user-input size parameter, the "size" of the probe used for calculations can be set to a radius of approximately 1.4 angstroms or less. Furthermore, a method for determining the surface-exposureable region using software for personal computers has been described by Pacios (Comput.Chem.18(4):377-386(1994); J.Mol.Model.1:46-53(1995)).Based on the above information, it is possible to select appropriate amino acid residues located on the surface of the polypeptide constituting the variant Fc region.

[0204] In certain embodiments, the polypeptide includes both a variant Fc region and an antigen-binding domain. In further embodiments, the antigen is a soluble antigen. In one embodiment, the antigen is present in the body fluid of the subject (e.g., plasma, interstitial fluid, lymph, ascites, and pleural fluid). The antigen may also be a membrane antigen.

[0205] In further embodiments, the antigen-binding activity of the antigen-binding domain changes according to ion concentration conditions. In one embodiment, ion concentration is not particularly limited and refers to hydrogen ion concentration (pH) or metal ion concentration. Here, metal ions refer to ions of Group I elements excluding hydrogen, such as alkali metals and copper group elements; Group II elements, such as alkaline earth metals and zinc group elements; Group III elements excluding boron; Group IV elements excluding carbon and silicon; Group VIII elements, such as iron group and platinum group elements; elements belonging to subgroup A of Groups V, VI, and VII; and metallic element ions such as antimony, bismuth, and polonium. In the present invention, metal ions include, for example, calcium ions, as described in International Publication No. 2012 / 073992 and International Publication No. 2013 / 125667. In one embodiment, “ion concentration conditions” may be conditions that focus on the difference in the biological behavior of the antigen-binding domain between low and high ion concentrations. Furthermore, the statement that "the antigen-binding activity of the antigen-binding domain changes depending on the ion concentration conditions" means that the antigen-binding activity of the antigen-binding domain changes between low and high ion concentrations (such antigen-binding domains are referred to herein as "ion concentration-dependent antigen-binding domains"). The antigen-binding activity of an antigen-binding domain under high ion concentration conditions may be higher (stronger) or lower (weaker) than under low ion concentration conditions. In one embodiment, an ion concentration-dependent antigen-binding domain (such as a pH-dependent antigen-binding domain or a calcium ion concentration-dependent antigen-binding domain) can be obtained by known methods, for example, those described in International Publication Nos. 2009 / 125825, 2012 / 073992, and 2013 / 046722.

[0206] In the present invention, the antigen-binding activity of the antigen-binding domain under high calcium ion concentration conditions may be higher than under low calcium ion concentration conditions. The high calcium ion concentration is not particularly limited, but may be a concentration selected from 100 μM to 10 mM, 200 μM to 5 mM, 400 μM to 3 mM, 200 μM to 2 mM, 400 μM to 1 mM, or 500 μM to 2.5 mM, and is preferably close to the in vivo plasma (blood) calcium ion concentration. On the other hand, the low calcium ion concentration is not particularly limited, but may be a concentration selected from 0.1 μM to 30 μM, 0.2 μM to 20 μM, 0.5 μM to 10 μM, 1 μM to 5 μM, or 2 μM to 4 μM, and is preferably close to the calcium ion concentration in the initial endosomes in vivo.

[0207] In one embodiment, the ratio of antigen-binding activity under low calcium ion concentration conditions to high calcium ion concentration conditions is not limited, but the ratio of the dissociation constant (KD) under low calcium ion concentration conditions to the KD under high calcium ion concentration conditions, i.e., KD(low calcium ion concentration conditions) / KD(high calcium ion concentration conditions), is 2 or more, 10 or more, or 40 or more. The upper limit of this ratio may be 400, 1000, or 10000, as long as such antigen-binding domains can be produced by techniques known to those skilled in the art. Alternatively, instead of KD, for example, the dissociation rate constant (kd) may be used. In this case, the ratio of the kd under low calcium ion concentration conditions to the kd under high calcium ion concentration conditions, i.e., kd(low calcium ion concentration conditions) / kd(high calcium ion concentration conditions), is 2 or more, 5 or more, 10 or more, or 30 or more. The upper limit of this ratio may be 50, 100, or 200, as long as antigen-binding domains can be produced based on common technical knowledge of those skilled in the art.

[0208] In the present invention, the antigen-binding activity of the antigen-binding domain under low hydrogen ion concentration (neutral pH) may be higher than under high hydrogen ion concentration (acidic pH). The acidic pH may be, for example, a pH selected from pH 4.0 to pH 6.5, a pH selected from pH 4.5 to pH 6.5, a pH selected from pH 5.0 to pH 6.5, or a pH selected from pH 5.5 to pH 6.5, and is preferably close to the in vivo pH of the initial endosome. The acidic pH may be, for example, pH 5.8 or pH 6.0. In a particular embodiment, the acidic pH is pH 5.8. The neutral pH may be, for example, a pH selected from pH 6.7 to pH 10.0, a pH selected from pH 6.7 to pH 9.5, a pH selected from pH 7.0 to pH 9.0, or a pH selected from pH 7.0 to pH 8.0, and is preferably close to the in vivo pH of plasma (blood). The neutral pH may be, for example, pH 7.4 or pH 7.0. In certain embodiments, the neutral pH is pH 7.4.

[0209] In one embodiment, the ratio of antigen-binding activity under acidic pH conditions to neutral pH conditions is not limited, but the ratio of the dissociation constant (KD) under acidic pH conditions to the KD under neutral pH conditions, i.e., KD(acidic pH conditions) / KD(neutral pH conditions), is 2 or more, 10 or more, or 40 or more. The upper limit of this ratio may be 400, 1000, or 10000, as long as such antigen-binding domains can be manufactured by techniques known to those skilled in the art. Alternatively, the dissociation rate constant (kd) can be used instead of KD. In this case, the ratio of kd under acidic pH conditions to kd under neutral pH conditions, i.e., kd(acidic pH conditions) / kd(neutral pH conditions), is 2 or more, 5 or more, 10 or more, or 30 or more. The upper limit of this ratio may be 50, 100, or 200, as long as antigen-binding domains can be manufactured based on common technical knowledge to those skilled in the art.

[0210] In one embodiment, for example, as described in International Publication No. 2009 / 125825, at least one amino acid residue is substituted with an amino acid residue having a side chain pKa of 4.0 to 8.0, and / or at least one amino acid having a side chain pKa of 4.0 to 8.0 is inserted into the antigen-binding domain. The amino acids can be substituted and / or inserted at any site, as long as the antigen-binding activity of the antigen-binding domain is weaker under acidic pH conditions than under neutral pH conditions compared to before substitution or insertion. If the antigen-binding domain has a variable region or CDR, the site may be within the variable region or CDR. The number of amino acids to be substituted or inserted can be appropriately determined by those skilled in the art, and the number may be one or more. By using amino acids having a side chain pKa of 4.0 to 8.0, the antigen-binding activity of the antigen-binding domain can be altered according to hydrogen ion concentration conditions. Examples of such amino acids include natural amino acids such as His(H) and Glu(E), as well as non-natural amino acids such as histidine analogs (US Patent Application Publication No. 2009 / 0035836), m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr (pKa 7.21), and 3,5-I2-Tyr (pKa 7.38) (Heyl et al., Bioorg. Med. Chem. 11(17):3761-3768 (2003)). Amino acids with side chain pKas of 6.0 to 7.0 can also be used, such as His(H).

[0211] In another embodiment, a preferred antigen-binding domain of the variant Fc region with increased pI is described, which can be obtained by the method described in International Publication No. 2016 / 125495 and International Publication No. 2017 / 046994.

[0212] In certain embodiments, the variant Fc region with increased pI includes at least two amino acid changes at at least two positions selected from the group consisting of: 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422, and 431 in EU numbering.

[0213] In further embodiments, the variant Fc region with increased pI comprises at least two amino acid changes at at least two positions selected from the group consisting of: 311, 341, 343, 384, 399, 400, 401, 402, and 413 in EU numbering.

[0214] In another embodiment, the present invention provides a polypeptide comprising a pI-imaged variant Fc region comprising any one of the following amino acid changes: (1) positions 311 and 341; (2) positions 311 and 343; (3) positions 311, 343 and 413; (4) positions 311, 384 and 413; (5) positions 311 and 399; (6) positions 311 and 401; (7) positions 311 and 413; (8) positions 400 and 413; (9) positions 401 and 413; and (10) positions 402 and 413.

[0215] In one embodiment, the present invention provides a polypeptide comprising a plurality of variant Fc regions having enhanced Fc-gamma-RIIb binding activity and increased pI, comprising at least three amino acid changes including the following: (a) Changes in at least one amino acid at at least one position selected from the group consisting of: EU numbering 231, 232, 233, 234, 235, 236, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334, and 396, and (b) Changes in at least two amino acids at at least two positions selected from the group consisting of: EU numbering 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422, and 431.

[0216] In one embodiment, the present invention provides a polypeptide comprising a variant Fc region (plural) having enhanced Fc-gamma-RIIb binding activity and increased pI, comprising at least three amino acid changes including: (a) at least one amino acid change at at least one position selected from the group consisting of: 231, 232, 235, 236, 239, 268, 295, 298, 326, 330, and 396 in EU numbering; and (b) at least two amino acid changes at at least two positions selected from the group consisting of: 311, 341, 343, 384, 399, 400, 401, 402, and 413 in EU numbering.

[0217] In another embodiment, the present invention provides polypeptides comprising a variant Fc region having enhanced Fc-gamma-RIIb binding activity and increased pI, comprising any one of the following amino acid changes (1) to (9): (1) positions 235, 236, 268, 295, 311, 326, 330 and 343 in EU numbering; (2) positions 236, 268, 295, 311, 326, 330 and 343; (3) positions 236, 268, 295, 311, 330 and 413; (4) positions 236, 268, 311, 330, 396 and 399; ( 5) Positions 236, 268, 311, 330 and 343; (6) Positions 236, 268, 311, 330, 343 and 413; (7) Positions 236, 268, 311, 330, 384 and 413; (8) Positions 236, 268, 311, 330 and 413; and (9) Positions 236, 268, 330, 396, 400 and 413.

[0218] In one embodiment, the present invention provides a polypeptide comprising a variant Fc region (plural) having enhanced Fc-gamma-RIIb binding activity and increased pI, comprising at least three amino acid changes including: (a) at least one amino acid change at at least one position selected from the group consisting of: 234, 238, 250, 264, 267, 307, and 330; and (b) at least two amino acid changes at at least two positions selected from the group consisting of: 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422, and 431 in EU numbering. In further embodiments, the polypeptide comprises at least two amino acid changes at at least two positions selected from the group consisting of: 311, 341, 343, 384, 399, 400, 401, 402, and 413 in EU numbering.

[0219] In another embodiment, the present invention provides polypeptides comprising a variant Fc region having enhanced Fc-gamma-RIIb binding activity and increased pI, comprising any one of the following amino acid changes (1) to (16): (1) positions 234, 238, 250, 264, 307, 311, 330 and 343 in EU numbering; (2) positions 234, 238, 250, 264, 307, 311, 330 and 413; (3) positions 234, 238, 250, 264, 267, 307, 311, 330 and 343; (4) positions 234, 238, 250, 264 , 267, 307, 311, 330 and 413; (5) Positions 234, 238, 250, 267, 307, 311, 330 and 343; (6) Positions 234, 238, 250, 267, 307, 311, 330 and 413; (7) Positions 234, 238, 250, 307, 311, 330 and 343; (8) Positions 234, 238, 250, 307, 311, 330 and 413; (9) Positions 238, 250, 264, 267, 307, 311, 330 and 343; (10) Positions 238, 250, 264, 267, 307, 311, 330 and 413; (11) Positions 238, 250, 264, 307, 311, 330 and 343; (12) Positions 238, 250, 264, 30 7, 311, 330 and 413; (13) positions 238, 250, 267, 307, 311, 330 and 343; (14) positions 238, 250, 267, 307, 311, 330 and 413; (15) positions 238, 250, 307, 311, 330 and 343; and (16) positions 238, 250, 307, 311, 330 and 413.

[0220] Furthermore, amino acid changes performed for other purposes(s) can be combined in the variant Fc region described herein. For example, amino acid substitutions that improve FcRn binding activity (Hinton et al., J.Immunol.176(1):346-356(2006); Dall'Acqua et al., J.Biol.Chem.281(33):23514-23524(2006); Petkova et al., Intl.Immunol.18(12):1759-1769(2006); Zalevsky et al., Nat.Biotechnol.28(2):157-159(2010); WO 2006 / 019447; WO 2006 / 053301; and WO 2009 / 086320), and amino acid substitutions that improve antibody heterogeneity or stability (WO 2009 / 041613) may be added. Alternatively, polypeptides having antigen-clearance-promoting properties as described in WO 2011 / 122011, WO 2012 / 132067, WO 2013 / 046704 or WO 2013 / 180201, polypeptides having specific binding properties to target tissues as described in WO 2013 / 180200, and polypeptides having repeated binding properties to multiple antigen molecules as described in WO 2009 / 125825, WO 2012 / 073992 or WO 2013 / 047752 can be combined with the variant Fc region described herein. Alternatively, amino acid changes disclosed in European Patent No. 1752471 and European Patent No. 1772465 can be combined at CH3 of the variant Fc region described herein for the purpose of conferring binding ability to other antigens. Alternatively, an amino acid change that reduces the constant region pI (International Publication No. 2012 / 016227) may be combined in the variant Fc region described herein for the purpose of increasing plasma retention. Alternatively, an amino acid change that increases the constant region pI (International Publication No. 2014 / 145159) may be combined in the variant Fc region described herein for the purpose of promoting uptake into cells.Alternatively, amino acid changes that increase the pI of the constant region (International Publication No. 2016 / 125495) can be combined in the variant Fc region described herein for the purpose of promoting the removal of target molecules from plasma. In one embodiment, such a modification may include, for example, a substitution at at least one position selected from the group consisting of 311, 343, 384, 399, 400, and 413 in EU numbering. In a further embodiment, such a substitution may be an amino acid substitution by Lys or Arg at each position.

[0221] Amino acid modifications that enhance human FcRn binding activity under acidic pH conditions can also be combined with the variant Fc region described herein. Specifically, such changes include, for example, the substitution of Leu for Met at position 428 in EU numbering and the substitution of Ser for Asn at position 434 (Zalevsky et al., Nat. Biotechnol. 28:157-159 (2010)); the substitution of Ala for Asn at position 434 (Deng et al., Metab. Dispos. 38(4):600-605 (2010)); the substitution of Tyr for Met at position 252, the substitution of Thr for Ser at position 254, and the substitution of Glu for Thr at position 256 (Dall'Acqua et al., J. Biol. Chem. 281:23514-23524 (2006)); the substitution of Gln for Thr at position 250, and the substitution of Leu for Met at position 428 (Hinton et al. al., J.Immunol. 176(1):346-356 (2006)); substitution of His for Asn at position 434 (Zheng et al., Clin.Pharmacol.Ther. 89(2):283-290 (2011), and WO 2010 / 106180, WO 2010 / 045193, WO 2009 / 058492, WO 2008 / 022152, WO 2006 / 050166, WO 2006 / 053301, WO 2006 / 031370, WO 2005 / 123780, WO 2005 / 047327, WO 2005 / 037867, WO These changes may include those described in 2004 / 035752 or WO 2002 / 060919. Examples of such changes include, for example, at least one change selected from the group consisting of the substitution of Leu for Met at position 428, the substitution of Ala for Asn at position 434, and the substitution of Thr for Tyr at position 436. These changes may further include the substitution of Arg for Gln at position 438 and / or the substitution of Glu for Ser at position 440 (International Publication 2016 / 125495).

[0222] Exemplary bispecific anti-CCL2 antibody One embodiment of the present invention is a bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a different second antigen-binding site that (specifically) binds to a different second epitope on human CCL2, A)i) The first antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 33, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 34, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 35, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 36, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 38, comprising a VL domain. Includes, ii) The second antigen-binding site described above is (a) a VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 43; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46, comprising the VL domain. including, or B)i) The first antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 33, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 34, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 35, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 36, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 38, comprising a VL domain. Includes, ii) The second antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 17, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 18, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 19, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 20, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 21, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 22, comprising a VL domain. including, or C)i) The first antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 33, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 34, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 35, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 36, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 38, comprising a VL domain. Includes, ii) The second antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 9, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 11, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 12, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 13, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 14, comprising the VL domain. including, or D)i) The first antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 17, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 18, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 19, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 20, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 21, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 22, comprising a VL domain. Includes, ii) The second antigen-binding site described above is (a) a VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 43; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46, comprising the VL domain. including, or E)i) The first antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 27, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 28, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 30, comprising a VL domain. Includes, ii) The second antigen-binding site described above is (a) a VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 43; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46, comprising the VL domain. including, or F)i) The first antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 49, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 50, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 51, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 52, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 53, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 54, comprising the VL domain. Includes, ii) The second antigen-binding site described above is (a) a VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 43; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46, comprising the VL domain. including, or G)i) The first antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 9, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 11, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 12, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 13, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 14, comprising the VL domain. Includes, ii) The second antigen-binding site is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 17, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 18, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 19, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 20, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 21, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 22, comprising a VL domain. including, or H)i) The first antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 9, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 11, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 12, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 13, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 14, comprising the VL domain. Includes, ii) The second antigen-binding site is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 27, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 28, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 30, comprising a VL domain. including, or I)i) The first antigen-binding site described above is (a) a VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 3; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 4, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 6, comprising a VL domain. Includes, ii) The second antigen-binding site is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 27, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 28, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 30, comprising a VL domain. It is a bispecific antibody that includes [specific component].

[0223] In one embodiment, the bispecific antibody comprises the Fc domain of a human IgG1 isotype.

[0224] In one embodiment, the bispecific antibody contains a constant heavy chain domain of a human IgG1 isotype.

[0225] In one embodiment, the in vivo clearance rate (ml / day / kg) of human CCL2 after administration of a bispecific antibody containing the constant heavy chain domain (or its Fc domain) of a human wild-type IgG1 isotype was at least twice as high (at least five times higher in one embodiment, at least ten times higher in one embodiment, and at least twenty times higher in one embodiment) when a pre-formed immune complex consisting of 20 mg / kg of each bispecific antibody and 0.1 mg / kg of human CCL2 was administered to FcRn transgenic mice at a single dose of 10 ml / kg, compared to the in vivo clearance rate (ml / day / kg) of human CCL2 after administration of a bispecific antibody containing the Fc gamma receptor silencing constant heavy chain domain (or its Fc domain) of a human IgG1 isotype including mutants L234A, L235A, and P329G.

[0226] One embodiment of the present invention is an isolated bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a different second epitope on human CCL2, i) The first antigen-binding site described above is A VH domain comprising the amino acid sequence of SEQ ID NO: 39, wherein (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 33, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 34, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 35; and A VL domain comprising the amino acid sequence of SEQ ID NO: 40, wherein (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 36, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 37, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 38. The antibody containing the antibody binds to the same epitope on CCL2, ii) The second antigen-binding site described above is A VH domain comprising the amino acid sequence of SEQ ID NO: 47, wherein (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 41, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 43; and A VL domain comprising the amino acid sequence of SEQ ID NO: 48, wherein (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 44, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46. It binds to the same epitope on CCL2 as the antibody containing it.

[0227] In one embodiment, the bispecific antibody comprises the Fc domain of a human IgG1 isotype.

[0228] In one embodiment, the bispecific antibody contains a constant heavy chain domain of a human IgG1 isotype.

[0229] In one embodiment, the in vivo clearance rate (ml / day / kg) of human CCL2 after administration of a bispecific antibody containing the constant heavy chain domain (or its Fc domain) of a human wild-type IgG1 isotype was at least 15 times higher, and particularly at least 20 times higher, when a pre-formed immune complex consisting of 20 mg / kg of each bispecific antibody and 0.1 mg / kg of human CCL2 was administered to FcRn transgenic mice in a single dose of 10 ml / kg, compared to the in vivo clearance rate (ml / day / kg) of human CCL2 after administration of a bispecific antibody containing the Fc gamma receptor silencing constant heavy chain domain (or its Fc domain) of a human IgG1 isotype including mutants L234A, L235A, and P329G.

[0230] One embodiment of the present invention is an isolated bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a different second epitope on human CCL2, i) The first antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) A VH domain comprising CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62, comprising the VL domain. Includes, ii) The second antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E, and the VH domain; and (d)X 1 is F or T, X 2 The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain Includes.

[0231] One embodiment of the present invention is an isolated bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a different second epitope on human CCL2, i) The first antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) CDR-H2 containing TANYAQKFQG, (d) CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59, (e) FR-H1 containing the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTF of SEQ ID NO: 63, (f) FR-H2 containing the amino acid sequence WVRQAPGQGLEWMG of SEQ ID NO: 64, (g) FR-H3 containing the amino acid sequence RVTITADESTSTAYMELSSLRSEDTAVY YCAR of SEQ ID NO: 65, and (g) FR-H4 containing the amino acid sequence WGQGTLVTVSS of SEQ ID NO: 66, the VH domain; and (h) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60; (i) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61; and (j) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62; (k) FR-L1 containing the amino acid sequence EIVLTQSPATLSLSPGERATLSC of SEQ ID NO: 67; (l) FR-L2 containing the amino acid sequence WYQQKPGQAPRLLIY of SEQ ID NO: 68; (m) FR-L3 containing the amino acid sequence GVPARFSGSGSGTDFTLTISSLEPEDFAVYYC of SEQ ID NO: 69; and (n) FR-L4 containing the amino acid sequence GQGTKVEIK of SEQ ID NO: 70, comprising the VL domain. Includes; ii) The second antigen-binding site described above is (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76; (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E; (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; (d) FR-H1 containing the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGLTIS of SEQ ID NO: 82; (e) FR-H2 containing the amino acid sequence WVRQAPGQGLEWMG of SEQ ID NO: 83; (f) FR-H3 containing the amino acid sequence RVTITADTSTSTAYMELSSLRSEDTAVYYCAR of SEQ ID NO: 84; and (g) FR-H4 containing the amino acid sequence WGQGTTVTVSS of SEQ ID NO: 85; and (h)X 1 is F or T, X 2 The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2VL domains comprising: (i) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80; (j) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R; (k) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC of SEQ ID NO: 86; (l) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIH of SEQ ID NO: 87; (m) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC of SEQ ID NO: 88; and (n) FR-L4 containing the amino acid sequence FGGGTKVEIK of SEQ ID NO: 89. Includes.

[0232] One embodiment of the present invention is an isolated bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a different second epitope on human CCL2, A)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 71; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 90; and VL domain containing the amino acid sequence of SEQ ID NO: 93 including, or B)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 71; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 91; and VL domain containing the amino acid sequence of SEQ ID NO: 93 including, or C)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 71; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 90; and VL domain containing the amino acid sequence of SEQ ID NO: 94 including, or D)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 72; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 90; and VL domain containing the amino acid sequence of SEQ ID NO: 94 including, or E)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 73; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 90; and VL domain containing the amino acid sequence of SEQ ID NO: 93 including, or F)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 73; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 90; and VL domain containing the amino acid sequence of SEQ ID NO: 94 including, or G)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 73; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 92; and VL domain containing the amino acid sequence of SEQ ID NO: 93 including, or H)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 73; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 91; and VL domain containing the amino acid sequence of SEQ ID NO: 93 including, or I)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 72; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 90; and VL domain containing the amino acid sequence of SEQ ID NO: 93 including, or J)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 72; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 92; and VL domain containing the amino acid sequence of SEQ ID NO: 93 including, or K)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 72; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 91; and VL domain containing the amino acid sequence of SEQ ID NO: 93 including, or L)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 74; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 90; and VL domain containing the amino acid sequence of SEQ ID NO: 93 including, or M)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 74; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 90; and VL domain containing the amino acid sequence of SEQ ID NO: 94 including, or N)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 74; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 92; and VL domain containing the amino acid sequence of SEQ ID NO: 93 including, or O)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 74; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 91; and VL domain containing the amino acid sequence of SEQ ID NO: 93 including, or P)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 71; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 92; and VL domain containing the amino acid sequence of SEQ ID NO: 93 It is a bispecific antibody that includes [specific component].

[0233] In one embodiment, the bispecific antibody comprises the Fc domain of a human IgG1 isotype.

[0234] In one embodiment, the bispecific antibody contains a constant heavy chain domain of a human IgG1 isotype.

[0235] One embodiment of the present invention is an isolated bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a different second epitope on human CCL2, i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 71; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 90; and VL domain containing the amino acid sequence of SEQ ID NO: 93 It is a bispecific antibody that includes [specific component].

[0236] One embodiment of the present invention is an isolated bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a different second epitope on human CCL2, i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 71; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 91; and VL domain containing the amino acid sequence of SEQ ID NO: 93 It is a bispecific antibody that includes [specific component].

[0237] One embodiment of the present invention is an isolated bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a different second epitope on human CCL2, i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 71; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 90; and VL domain containing the amino acid sequence of SEQ ID NO: 94 It is a bispecific antibody that includes [specific component].

[0238] One embodiment of the present invention is an isolated bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a different second epitope on human CCL2, i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 72; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 90; and VL domain containing the amino acid sequence of SEQ ID NO: 94 It is a bispecific antibody that includes [specific component].

[0239] In one embodiment, the bispecific antibody comprises the Fc domain of a human IgG1 isotype.

[0240] In one embodiment, the bispecific antibody comprises a constant heavy chain domain of a human IgG1 isotype. One embodiment of the present invention provides an isolated bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a different second epitope on human CCL2, A)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 71, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I, and (b) X 1 V is X 2 P is X 3 The amino acid sequence GX of sequence number 58 is H. 1 IX 2 IFX 3(c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 90, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 93, and (d)X 1 F is X 2 The amino acid sequence KAX of sequence number 79 is R. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W, and the VL domain sequence including, or B)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 71, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I, and (b) X 1 V is X 2 P is X 3 The amino acid sequence GX of sequence number 58 is H. 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 91, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 93, and (d)X 1 F is X 2 The amino acid sequence KAX of sequence number 79 is R. 1EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W, and the VL domain sequence including, or C)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 71, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 90, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 94, and (d)X 1 is F or T, X 2 The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain sequence including, or D)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 72, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 90, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 94, and (d)X 1 is F or T, X 2 The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain sequence including, or E)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 73, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 90, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 93, and (d)X 1 is F or T, X 2The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain sequence including, or F)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 73, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 90, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 94, and (d)X 1 is F or T, X 2 The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain sequence including, or G)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 73, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 92, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 93, and (d)X 1 is F or T, X 2 The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain sequence including, or H)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 73, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 91, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 93, and (d)X 1 is F or T, X 2The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain sequence including, or I)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 72, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 90, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 93, and (d)X 1 is F or T, X 2 The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain sequence including, or J)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 72, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 92, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 93, and (d)X 1 is F or T, X 2 The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain sequence including, or K)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 72, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 91, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 93, and (d)X 1 is F or T, X 2The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain sequence including, or L)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 74, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 90, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 93, and (d)X 1 is F or T, X 2 The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain sequence including, or M)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 74, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 90, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 94, and (d)X 1 is F or T, X 2 The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain sequence including, or N)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 74, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 92, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 93, and (d)X 1 is F or T, X 2The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain sequence including, or O)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 74, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 91, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 93, and (d)X 1 is F or T, X 2 The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain sequence including, or P)i) The first antigen-binding site described above is A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 71, and (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, and (b) X 1 is V, I or H, and X 2 is P or H, X 3 The amino acid sequence GX of SEQ ID NO: 58, where H or G 1 IX 2 IFX 3 (c) VH domain sequences including CDR-H2 containing TANYAQKFQG, and CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, comprising (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Includes, ii) The second antigen-binding site is VH domain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 92, comprising (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; and A VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of sequence number 93, and (d)X 1 is F or T, X 2 The amino acid sequence KAX of SEQ ID NO: 79, where R or L is present. 1 EDIYNRX 2 (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R, and the VL domain It is a bispecific antibody that includes [specific component].

[0241] In one embodiment, the bispecific antibody comprises the Fc domain of a human IgG1 isotype.

[0242] In one embodiment, the bispecific antibody contains a constant heavy chain domain of a human IgG1 isotype.

[0243] In one embodiment, the bispecific antibody described herein is i) Block the binding of CCL2 to its receptor CCR2 in vitro (reporter assay, IC) 50 =0.5nM); and / or ii) Inhibit CCL2-mediated chemotaxis of myeloid cells in vitro (IC 50 =1.5nM); and / or iii) Cross-reactivity with cynomolgus monkeys and human CCL2.

[0244] In one embodiment, the bispecific antibody described herein is not cross-reactive to other human CCL homologs, and in particular, exhibits 100-fold less binding to other CCL homologs (selected from the group CCL8, CCL7, and CCL13) compared to binding to CCL2.

[0245] In one embodiment, the bispecific antibody described herein binds to first and second epitopes on human CCL2 in an ion-dependent manner.

[0246] In one embodiment, the bispecific antibody described herein binds to human CCL2 in a pH-dependent manner, with both the first and second antigen-binding sites binding to CCL2 with higher affinity at neutral pH than at acidic pH.

[0247] In one embodiment, the bispecific antibody described herein binds to human CCL2 at pH 7.4 with 10 times higher affinity than at pH 5.8.

[0248] In one embodiment, the bispecific antibody described herein comprises two IgG1 heavy chain constant domains (or their Fc domains) containing the following mutations (EU numbering) (independently or in addition to the above mutations). i) S354C and T366W in one of the heavy chain constant domains ii) Y349C, T366S, L368A, Y407V in the other heavy chain constant domain

[0249] In one embodiment, the bispecific antibody comprises the Fc domain of a human IgG1 isotype.

[0250] In one embodiment, the bispecific antibody comprises a constant heavy chain domain of a human IgG1 isotype. In one embodiment, the bispecific antibody described herein comprises a human IgG1 heavy chain constant domain (or its Fc domain) comprising one or more of the following mutations (Kabat EU numbering). i) Q311R and / or P343R (suitable for increasing pI to enhance antigen uptake); and / or ii) L234Y, L235W, G236N, P238D, T250V, V264I, H268D, Q295L, T307P, K326T and / or A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and / or iii) M428L, N434A and / or Y436T (suitable for increasing affinity to FcRn for a longer plasma half-life); and / or iv) Q438R and / or S440E (suitable for inhibiting rheumatoid factor binding).

[0251] In one embodiment, the bispecific antibody described herein comprises a human IgG1 heavy chain constant domain (or its Fc domain) containing one or more of the following mutations (Kabat EU numbering). i) Q311R, and / or P343R (suitable for increasing pI to enhance antigen uptake); and / or ii) L235W, G236N, H268D, Q295L, K326T and / or A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and / or iii) N434A (suitable for increasing affinity to FcRn for a longer plasma half-life); and / or iv) Q438R and / or S440E (suitable for inhibiting rheumatoid factor binding).

[0252] In one embodiment, the bispecific antibody described herein comprises a human IgG1 heavy chain constant domain (or its Fc domain) containing the following mutation (Kabat EU numbering). i) Q311R and P343R (suitable for increasing pI to enhance antigen uptake); and ii) L235W, G236N, H268D, Q295L, K326T and A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and iii) N434A (suitable for increasing affinity to FcRn for a longer plasma half-life); and iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

[0253] In one embodiment, the bispecific antibody described herein comprises a human IgG1 heavy chain constant domain (or its Fc domain) containing the following mutation (Kabat EU numbering). i) Q311R and P343R (suitable for increasing pI to enhance antigen uptake); and ii) N434A (suitable for increasing affinity to FcRn for a longer plasma half-life); and iii) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

[0254] In one embodiment, the bispecific antibody described herein comprises a human IgG1 heavy chain constant domain (or its Fc domain) containing the following mutation (Kabat EU numbering). Q311R and P343R (suitable for increasing pI to enhance antigen uptake).

[0255] In one embodiment, the bispecific antibody described herein comprises a human IgG1 heavy chain constant domain (or its Fc domain) containing one or more of the following mutations (Kabat EU numbering). i) Q311R and / or P343R (suitable for increasing pI to enhance antigen uptake); and / or ii) L234Y, P238D, T250V, V264I, T307P and / or A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and / or iii) M428L, N434A and / or Y436T (suitable for increasing affinity to FcRn for a longer plasma half-life); and / or iv) Q438R and / or S440E (suitable for inhibiting rheumatoid factor binding).

[0256] In one embodiment, the bispecific antibody described herein comprises a human IgG1 heavy chain constant domain (or its Fc domain) containing one or more of the following mutations (Kabat EU numbering). i) Q311R and P343R (suitable for increasing pI to enhance antigen uptake); and ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and iii) M428L, N434A and Y436T (suitable for increasing affinity to FcRn for a longer plasma half-life); and iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

[0257] In one embodiment, the bispecific antibody described herein comprises a human IgG1 heavy chain constant domain (or its Fc domain) containing one or more of the following mutations (Kabat EU numbering). i) Q311R and P343R (suitable for increasing pI to enhance antigen uptake); and ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and iii) N434A and (suitable for increasing affinity for FcRn due to a longer plasma half-life); and iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

[0258] One embodiment of the present invention is an isolated bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a different second epitope on human CCL2, A)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 71; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 90; and VL domain containing the amino acid sequence of SEQ ID NO: 93 including, or B)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 71; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 91; and VL domain containing the amino acid sequence of SEQ ID NO: 93 including, or C)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 71; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 90; and VL domain containing the amino acid sequence of SEQ ID NO: 94 including, or D)i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 72; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 90; and VL domain containing the amino acid sequence of SEQ ID NO: 94 Includes, The above bispecific antibody is a full-length antibody of a human IgG isotype (preferably human IgG1 isotype) having the following characteristics: a) below each i) the first light chain and the first heavy chain of the first antibody containing the first antigen-binding site; and b) A second light chain and a second heavy chain of a second antibody, each containing the second antigen-binding site described above under ii), wherein the variable domains VL and VH in the second light chain and second heavy chain of the second antibody are substituted for each other; In the constant domain CL of the first light chain under a), the amino acid at position 124 is substituted with lysine (K) (numbering follows Kabat), and the amino acid at position 123 is substituted with lysine (K) (numbering follows Kabat), In the constant domain CH1 of the first heavy chain below a), the amino acid at position 147 is substituted with glutamic acid (E) (numbering follows the Kabat EU index), and the amino acid at position 213 is substituted with glutamic acid (E) (numbering follows the Kabat EU index).

[0259] One embodiment of the present invention is an isolated bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a different second epitope on human CCL2, i) The first antigen-binding site described above is VH domain containing the amino acid sequence of SEQ ID NO: 71; and VL domain containing the amino acid sequence of SEQ ID NO: 75 Includes, ii) The second antigen-binding site is VH domain containing the amino acid sequence of SEQ ID NO: 91; and VL domain containing the amino acid sequence of SEQ ID NO: 93 Includes, The above bispecific antibody is a (full-length) antibody having the following properties: a)i) below the first kappa or lambda light chain and the first heavy chain of the IgG1 isotype containing the first antigen-binding site; and b)ii) a second kappa or lambda light chain and a second IgG1 heavy chain of an IgG1 isotype containing the second antigen-binding site, wherein the variable domains VL and VH in the second light chain and second heavy chain of the second antibody are substituted for each other; In the constant domain CL of the first light chain under a), the amino acid at position 124 is substituted with lysine (K) (numbering follows Kabat), and the amino acid at position 123 is substituted with lysine (K) (numbering follows Kabat), In the constant domain CH1 of the first heavy chain below a), the amino acid at position 147 is substituted with glutamic acid (E) (numbering follows the Kabat EU index), and the amino acid at position 213 is substituted with glutamic acid (E) (numbering follows the Kabat EU index).

[0260] In one embodiment, such a bispecific antibody includes the following mutation (Kabat EU numbering): i) S354C and T366W in one of the heavy chain constant domains ii) Y349C, T366S, L368A, Y407V in the other heavy chain constant domain

[0261] In one embodiment, such a bispecific antibody further includes the following mutations (Kabat EU numbering): i) Q311R and P343R (suitable for increasing pI to enhance antigen uptake); and ii) L235W, G236N, H268D, Q295L, K326T and A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and iii) N434A (suitable for increasing affinity to FcRn for a longer plasma half-life); and iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

[0262] In one alternative embodiment, such a bispecific antibody further includes the following mutations (Kabat EU numbering): i) Q311R and P343R (suitable for increasing pI to enhance antigen uptake); and ii) N434A (suitable for increasing affinity to FcRn for a longer plasma half-life); and iii) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

[0263] In one alternative embodiment, such a bispecific antibody further includes the following mutations (Kabat EU numbering): Q311R and P343R (suitable for increasing pI to enhance antigen uptake).

[0264] In one alternative embodiment, such a bispecific antibody further includes the following mutations (Kabat EU numbering): i) Q311R and P343R (suitable for increasing pI to enhance antigen uptake); and ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and iii) M428L, N434A and Y436T (suitable for increasing affinity to FcRn for a longer plasma half-life); and iv) Q438R and S440E are suitable for inhibiting rheumatoid factor binding.

[0265] In one alternative embodiment, such a bispecific antibody further includes the following mutations (Kabat EU numbering): i) Q311R and P343R (suitable for increasing pI to enhance antigen uptake); and ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and iii) N434A and (suitable for increasing affinity for FcRn due to a longer plasma half-life); and iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

[0266] A specific embodiment of the present invention is a (isolated) bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, the bispecific antibody comprising a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 112, a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 113, a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 114, and a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 115.

[0267] A specific embodiment of the present invention is a (isolated) bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, the bispecific antibody comprising a polypeptide containing the amino acid sequence of SEQ ID NO: 112, a polypeptide containing the amino acid sequence of SEQ ID NO: 113, a polypeptide containing the amino acid sequence of SEQ ID NO: 114, and a polypeptide containing the amino acid sequence of SEQ ID NO: 115.

[0268] A specific embodiment of the present invention is a (isolated) bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, the bispecific antibody comprising a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 116, a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 117, a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 118, and a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 119.

[0269] A specific embodiment of the present invention is a (isolated) bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, the bispecific antibody comprising a polypeptide containing the amino acid sequence of SEQ ID NO: 116, a polypeptide containing the amino acid sequence of SEQ ID NO: 117, a polypeptide containing the amino acid sequence of SEQ ID NO: 118, and a polypeptide containing the amino acid sequence of SEQ ID NO: 119.

[0270] A specific embodiment of the present invention is a (isolated) bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, the bispecific antibody comprising a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 120, a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 121, a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 122, and a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 123.

[0271] A specific embodiment of the present invention is a (isolated) bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, the bispecific antibody comprising a polypeptide containing the amino acid sequence of SEQ ID NO: 120, a polypeptide containing the amino acid sequence of SEQ ID NO: 121, a polypeptide containing the amino acid sequence of SEQ ID NO: 122, and a polypeptide containing the amino acid sequence of SEQ ID NO: 123.

[0272] A specific embodiment of the present invention is a (isolated) bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, the bispecific antibody comprising a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 120, a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 121, a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 122, and a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 123.

[0273] A specific embodiment of the present invention is a (isolated) bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, the bispecific antibody comprising a polypeptide containing the amino acid sequence of SEQ ID NO: 120, a polypeptide containing the amino acid sequence of SEQ ID NO: 121, a polypeptide containing the amino acid sequence of SEQ ID NO: 122, and a polypeptide containing the amino acid sequence of SEQ ID NO: 123.

[0274] A specific embodiment of the present invention is a (isolated) bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, the bispecific antibody comprising a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 155, a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 156, a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 157, and a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 158.

[0275] A specific embodiment of the present invention is a (isolated) bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, the bispecific antibody comprising a polypeptide containing the amino acid sequence of SEQ ID NO: 155, a polypeptide containing the amino acid sequence of SEQ ID NO: 156, a polypeptide containing the amino acid sequence of SEQ ID NO: 157, and a polypeptide containing the amino acid sequence of SEQ ID NO: 158.

[0276] A specific embodiment of the present invention is a (isolated) bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, the bispecific antibody comprising a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 159, a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 160, a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 161, and a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 162.

[0277] A specific embodiment of the present invention is a (isolated) bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, the bispecific antibody comprising a polypeptide containing the amino acid sequence of SEQ ID NO: 159, a polypeptide containing the amino acid sequence of SEQ ID NO: 160, a polypeptide containing the amino acid sequence of SEQ ID NO: 161, and a polypeptide containing the amino acid sequence of SEQ ID NO: 162.

[0278] A specific embodiment of the present invention is a (isolated) bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, the bispecific antibody comprising a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 163, a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 164, a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 165, and a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 166.

[0279] A specific embodiment of the present invention is a (isolated) bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, the bispecific antibody comprising a polypeptide containing the amino acid sequence of SEQ ID NO: 163, a polypeptide containing the amino acid sequence of SEQ ID NO: 164, a polypeptide containing the amino acid sequence of SEQ ID NO: 165, and a polypeptide containing the amino acid sequence of SEQ ID NO: 166.

[0280] A specific embodiment of the present invention is a (isolated) bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, the bispecific antibody comprising a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 167, a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 168, a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 169, and a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 170.

[0281] A specific embodiment of the present invention is a (isolated) bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, the bispecific antibody comprising a polypeptide containing the amino acid sequence of SEQ ID NO: 167, a polypeptide containing the amino acid sequence of SEQ ID NO: 168, a polypeptide containing the amino acid sequence of SEQ ID NO: 169, and a polypeptide containing the amino acid sequence of SEQ ID NO: 170.

[0282] A specific embodiment of the present invention is a (isolated) bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, the bispecific antibody comprising a polypeptide having an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 171, a polypeptide having an amino acid sequence that is at least 98% or 99% ident...

Claims

1. A bispecific antibody comprising a first antigen-binding site that binds to a first epitope on human CCL2 and a second antigen-binding site that binds to a second epitope on human CCL2, i) The first antigen-binding site is (a) CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I; (b) CDR-H2 containing the amino acid sequence GX1IX2IFX3TANYAQKFQG of SEQ ID NO: 58, where X1 is V, X2 is P, and X3 is H; and (c) CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59, comprising a VH domain; and (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE of SEQ ID NO: 61; and (f) a VL domain containing CDR-L3 containing the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62; Includes, ii) The second antigen-binding site is (a) CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X is D, and (c) CDR-H3 containing the amino acid sequence GVGFFFXH of SEQ ID NO: 78, where X is E; and VH domains; (d) CDR-L1 containing the amino acid sequence KAX1EDIYNRX2A of SEQ ID NO: 79, where X1 is F and X2 is R; (e) CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80; and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W, comprising the VL domain. Includes, The bispecific antibody comprises a first polypeptide having an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 159; a second polypeptide having an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 160; a third polypeptide having an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 161; and a fourth polypeptide having an amino acid sequence having at least 98% sequence identity with SEQ ID NO:

162.

2. The bispecific antibody according to claim 1, comprising: a first polypeptide having an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 159; a second polypeptide having an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 160; a third polypeptide having an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 161; and a fourth polypeptide having an amino acid sequence having at least 99% sequence identity with SEQ ID NO:

162.

3. A bispecific antibody according to claim 1 or 2, comprising a first polypeptide having the amino acid sequence of SEQ ID NO: 159; a second polypeptide having the amino acid sequence of SEQ ID NO: 160; a third polypeptide having the amino acid sequence of SEQ ID NO: 161; and a fourth polypeptide having the amino acid sequence of SEQ ID NO:

162.

4. i) Blocking the binding of CCL2 to its receptor CCR2 in vitro (reporter assay, IC50 = 0.5 nM); and / or ii) Inhibit CCL2-mediated chemotaxis of myeloid cells in vitro (IC50 = 1.5 nM); and / or iii) A bispecific antibody according to any one of claims 1 to 3, which is cross-reactive to cyno and human CCL2.

5. A bispecific antibody according to any one of claims 1 to 4, which is not cross-reactive to other CCL homologs.

6. A bispecific antibody according to any one of claims 1 to 5, which binds to human CCL2 at pH 7.4 with 10 times higher affinity than at pH 5.

8.

7. The following mutations (Kabat EU numbering): i) Q311R and / or P343R; and / or ii) L234Y, L235W, G236N, P238D, T250V, V264I, H268D, Q295L, T307P, K326T and / or A330K; and / or iii) M428L, N434A and / or Y436T; and / or iv) Q438R and / or S440E A bispecific antibody according to any one of claims 1 to 6, comprising a human IgG1 heavy chain constant domain containing one or more of the following.

8. An isolated nucleic acid encoding the antibody according to any one of claims 1 to 7.

9. A host cell comprising the nucleic acid described in Claim 8.

10. A method for producing an antibody, comprising culturing the host cells described in claim 9 so as to produce the antibody.

11. The method according to claim 10, further comprising recovering the antibody from the host cell.

12. A pharmaceutical formulation comprising a bispecific antibody according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

13. A bispecific antibody according to any one of claims 1 to 7, for use as a pharmaceutical.

14. A bispecific antibody according to any one of claims 1 to 7, for use in the treatment of cancer.

15. A bispecific antibody according to any one of claims 1 to 7, for use in the treatment of inflammatory diseases or autoimmune diseases.

16. Use of a bispecific antibody according to any one of claims 1 to 7 in the manufacture of a pharmaceutical product.

17. The use according to claim 16, wherein the pharmaceutical is for the treatment of cancer.

18. The use according to claim 16, wherein the pharmaceutical is for the treatment of an inflammatory disease or an autoimmune disease.

19. A pharmaceutical for treating an individual having cancer, comprising an effective amount of a bispecific antibody according to any one of claims 1 to 7.

20. A pharmaceutical for treating an individual having an inflammatory disease or an autoimmune disease, comprising an effective amount of a bispecific antibody according to any one of claims 1 to 7.

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