HK40077249A

HK40077249A - Bispecific anti-ccl2 antibodies

Info

Publication number: HK40077249A
Application number: HK62023066588.2A
Authority: HK
Inventors: 冯舒; J·费歇尔; S·K·甘; G·乔治; M·格茨; W·S·A·何; A·约瑟夫; G·乔丹; H·凯滕伯格; R·A·林; M·马捷特; J·马利肯; V·伦扎; M·舍费尔; T·施洛托埃; G·蒂芬太勒; M·维耶特
Original assignee: 豪夫迈·罗氏有限公司
Priority date: 2019-12-18
Filing date: 2020-12-16
Publication date: 2023-03-03

Description

Bispecific anti-CCL 2 antibodies

Technical Field

The present invention relates to bispecific anti-CCL 2 antibodies that bind to two different epitopes on human CCL2, pharmaceutical compositions thereof, their manufacture, and use as medicaments for the treatment of cancer, inflammatory diseases, autoimmune diseases, and ophthalmic diseases.

Background

The CCL2/CCR2 axis is the primary mediator that recruits immature myeloid cells into tumors. CCL2 is overexpressed by malignant cells and binds to the extracellular matrix (ECM), establishing a chemotactic gradient. Once they reach the tumor, myeloid-derived suppressor cells (MDSCs) in turn suppress the initiation of anti-tumor T cell responses by secreting/upregulating anti-inflammatory cytokines/receptors, contributing to the original tumor environment. In this way, MDSCs can reduce or even impair the efficacy of any T cell activation therapy (Meyer et al, 2014). Thus, specific inhibition of recruitment of these immature myeloid cells will improve the efficacy of checkpoint inhibitors, T cell bispecific antibodies (TCBs), or other Cancer Immunotherapies (CITs). In addition, CCL2 is also associated with promoting angiogenesis, metastasis, and tumor growth, suggesting that neutralization of CCL2 may contribute to a variety of anti-tumor interventions.

In contrast to its receptor, targeting CCL2 will specifically inhibit the undesired CCL2 mediated effects, thereby avoiding those signals that might be emitted by the same receptor (CCR2) 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.

CCL2 is a preferred antibody target in several studies clinically directed to neutralising elevated levels caused by different 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.2015Dec; 46 (6): 1740-50), diabetic nephropathy (Menne et al, Nephrol Dial Transplant (2017) 32: 307 Apr 315) and Cancer (Sandhu et al, Cancer Chemothermal Phacol.2013 Apr; 71 (4): 1041-50). However, its high synthesis rate and the observed high in vivo antibody-antigen dissociation constant (KD) have been demonstrated to be major obstacles that prevent conventional antibodies from inhibiting free CCL2 at clinically feasible doses (Fetterly et al, J Clin Pharmacol.2013 Oct; 53(10): 1020-7).

Neutralization of CCL2 appears to be more significantly associated with patients with elevated serum CCL2 levels, which has been observed in various types of cancers, such as Breast Cancer (BC), ovarian cancer (OvCa), colorectal cancer (CRC), pancreatic cancer, and prostate cancer. However, even though patients within these indications did not exhibit such serology but had tumors that were highly infiltrated by myeloid immune cells, patients may benefit from this novel treatment due to the multiple roles CCL2 plays in the tumor environment as described above.

Igawa et al, Immunological Reviews 270(2016)132-151, describe a Sweeping (Sweeping) technique in which the resulting antibody carries pH-dependent CDRs (for antibody-antigen dissociation in acidic endosomes, leading to antigen degradation) and an engineered Fc portion with an optimized isoelectric point (pI) and enhanced binding to Fc γ RIIb (favoring cellular uptake of the immune complex) and moderate affinity to neonatal Fc receptors to maintain acceptable pharmacokinetic profiles.

Disclosure of Invention

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

One embodiment of the invention is a bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second, different antigen-binding site that (specifically) binds to a second epitope on human CCL2,

Wherein the content of the first and second substances,

A) i) the first antigen binding site comprises

A VH domain comprising: (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: (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; and is

ii) the second antigen binding site comprises

A VH domain comprising: (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: (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;

or

B) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

A VH domain comprising: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO 17; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO. 18; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO. 19;

and a VL domain comprising: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO. 20; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO 21; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO. 22;

or

C) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

A VH domain comprising: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO 9; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO. 10; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO. 11;

and a VL domain comprising: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO. 12; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO 13; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO. 14;

or

D) i) the first antigen binding site comprises

and a VL domain comprising: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO. 20; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO 21; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO. 22; and is

ii) the second antigen binding site comprises

or

E) i) the first antigen binding site comprises

A VH domain comprising: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO. 25; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO. 26; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27;

and a VL domain comprising: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO 28; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 29; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30; and is

ii) the second antigen binding site comprises

A VH domain comprising: (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: (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;

Or

F) i) the first antigen binding site comprises

A VH domain comprising: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO. 49; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO 50; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 51;

and a VL domain comprising: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO 52; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO 53; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; and is

ii) the second antigen binding site comprises

or

G) i) the first antigen binding site comprises

And a VL domain comprising: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO. 12; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO 13; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO. 14; and is

ii) the second antigen binding site comprises

or

H) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

and a VL domain comprising: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO 28; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 29; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30;

or

I) i) the first antigen binding site comprises

A VH domain comprising: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO 1; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO. 2; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO. 3;

and a VL domain comprising: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO. 4; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO 5; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO 6; and is

ii) the second antigen binding site comprises

And a VL domain comprising: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO 28; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 29; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

In one embodiment, the bispecific antibody comprises an Fc domain of a human IgG isotype.

In one embodiment, the bispecific antibody comprises an Fc domain of the human IgG1 isotype.

In one embodiment, the bispecific antibody comprises a constant heavy chain domain of a human IgG isotype.

In one embodiment, the bispecific antibody comprises a constant heavy chain domain of human IgG1 isotype.

In one embodiment, when a pre-formed immune complex consisting of 20mg/kg of each bispecific antibody and 0.1mg/kg of human CCL2 is administered to an FcRn transgenic mouse at a single dose of 10ml/kg, the in vivo clearance (ml/day/kg) of human CCL2 is at least two-fold higher (in one embodiment, at least 5-fold higher, in one embodiment, at least 10-fold higher, in one embodiment, at least 20-fold higher) after administration of the bispecific antibody comprising an fcy receptor silencing constant heavy chain domain (or Fc domain thereof) of the human IgG1 isotype comprising the mutations L234A, L235A, P329G (Kabat EU numbering) compared to the in vivo clearance (ml/day/kg) of human CCL2 after administration of the bispecific antibody comprising an fcy receptor silencing constant heavy chain domain (or Fc domain thereof) of the human IgG1 isotype of human wild-type IgG 1.

One embodiment of the 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,

wherein, the first and the second end of the pipe are connected with each other,

i) the first antigen binding site binds to the same epitope on CCL2 as does an antibody comprising

39, wherein the VH domain comprises an amino acid sequence of SEQ ID NO: (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 the VL domain comprises: (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; and is

ii) said second antigen binding site binds to the same epitope on CCL2 as does an antibody comprising

47, wherein the VH domain comprises: (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 the VL domain comprises: (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.

In one embodiment, when a preformed immune complex consisting of 20mg/kg of each bispecific antibody and 0.1mg/kg of human CCL2 is administered to an FcRn transgenic mouse at a single dose of 10ml/kg, the in vivo clearance (ml/day/kg) of human CCL 389 2 is at least 15-fold higher, in particular at least 20-fold higher, after administration of the bispecific antibody comprising an fcgamma receptor silencing constant heavy chain domain of human IgG1 isotype (or an Fc domain thereof) comprising mutations L234A, L235A, P329G (Kabat EU numbering) compared to the in vivo clearance (ml/day/kg) of human CCL2 after administration of the bispecific antibody comprising an fcgamma receptor silencing constant heavy chain domain of human IgG1 isotype (or an Fc domain thereof) comprising mutations L234A, L235, P32 (Kabat EU numbering).

i) the first antigen binding site comprises

A VH domain comprising: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO 57, wherein X is I or T; (b) CDR-H2 comprising the amino acid sequence GX of SEQ ID NO:58 ¹ IX ² IFX ³ TANYAQKFQG, wherein X ¹ Is V, I or H, X ² Is P or H, and X ³ Is H or G; and (c) CDR-H3 comprising amino acid sequence YDAHYGELDF of SEQ ID NO: 59;

and

a VL domain comprising: (d) CDR-L1 comprising amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 61; and (f) CDR-L3 comprising amino acid sequence HQYIHLHSFT of SEQ ID NO: 62;

and is

ii) the second antigen binding site comprises

A VH domain 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, wherein X is D or E; and (c) CDR-H3 comprising the amino acid sequence GVGFFXH of SEQ ID NO. 78, wherein X is D or E;

and

a VL domain comprising: (d) CDR-L1 comprising the amino acid sequence KAX of SEQ ID NO:79 ¹ EDIYNRX ² A, wherein, X ¹ Is F or T and X ² Is R or L; (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, wherein X is W or R.

i) the first antigen binding site comprises

A VH domain comprising: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO 57, wherein X is I or T; (b) CDR-H2 comprising the amino acid sequence GX of SEQ ID NO:58 ¹ IX ² FX ³ TANYAQKFQG, wherein X ¹ Is V, I or H, X ² Is P or H, and X ³ Is H or G; (c) CDR-H3 comprising amino acid sequence YDAHYGELDF of SEQ ID NO: 59; (d) FR-H1 comprising the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTF of SEQ ID NO: 63; (e) FR-H2 comprising the amino acid sequence WVRQAPGQGLEWMG of SEQ ID NO 64; (f) FR-H3 comprising the amino acid sequence RVTITADESTSTAYMELSSLRSEDTAVYYCAR of SEQ ID NO: 65; and (g) FR-H4 comprising the amino acid sequence WGQGTLVTVSS of SEQ ID NO: 66;

and

a VL domain comprising: (h) CDR-L1 comprising amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60; (i) CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 61; and (j) CDR-L3 comprising amino acid sequence HQYIHLHSFT of SEQ ID NO: 62; (k) FR-L1 comprising the amino acid sequence EIVLTQSPATLSLSPGERATLSC of SEQ ID NO: 67; (l) FR-L2 comprising the amino acid sequence WYQQKPGQAPRLLIY of SEQ ID NO: 68; (m) FR-L3 comprising the amino acid sequence GVPARFSGSGSGTDFTLTISSLEPEDFAVYYC of SEQ ID NO: 69; and (n) FR-L4 comprising amino acid sequence GQGTKVEIK of SEQ ID NO: 70;

And is provided with

ii) the second antigen binding site comprises

A VH domain 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, wherein X is D or E; (c) CDR-H3 comprising the amino acid sequence GVGFFXH of SEQ ID NO. 78 wherein X is D or E; (d) FR-H1 comprising the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGLTIS of SEQ ID NO: 82; (e) FR-H2 comprising the amino acid sequence WVRQAPGQGLEWMG of SEQ ID NO 83; (f) FR-H3 comprising the amino acid sequence RVTITADTSTSTAYMELSSLRSEDTAVYYCAR of SEQ ID NO: 84; and (g) FR-H4 comprising amino acid sequence WGQGTTVTVSS of SEQ ID NO: 85;

and

a VL domain comprising: (h) CDR-L1 comprising the amino acid sequence KAX of SEQ ID NO:79 ¹ EDIYNRX ² A, wherein, X ¹ Is F or T and X ² Is R or L; (i) CDR-L2 comprising the amino acid sequence GATSLEH of SEQ ID NO. 80; (j) CDR-L3 comprising the amino acid sequence QQFXSAPYT of SEQ ID NO. 81, wherein X is W or R; (k) FR-L1 comprising the amino acid sequence DIQMTQSPSSLSASVGDRVTITC of SEQ ID NO 86; (l) FR-L2 comprising the amino acid sequence WYQQKPGKAPKLLIH of SEQ ID NO: 87; (m) FR-L3 comprising the amino acid sequence GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC of SEQ ID NO: 88; and (n) FR-L4 comprising the amino acid sequence FGGGTKVEIK of SEQ ID NO: 89.

wherein the content of the first and second substances,

A) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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

B) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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

C) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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 94;

or alternatively

D) 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 is

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 94;

or

E) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 73;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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

F) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 73;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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 94;

or alternatively

G) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 73;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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

H) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 73;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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

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 is

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 alternatively

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 is

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

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 is

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

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 is

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 alternatively

M) 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 is

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 94;

or

N) 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 is

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

O) i) said 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 is

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

P) i) said first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is provided with

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.

wherein the content of the first and second substances,

A) i) the first antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO 71, wherein the VH domain comprises: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO 57, wherein X is I; (b) CDR-H2 comprising the amino acid sequence GX of SEQ ID NO:58 ¹ IX ² IFX ³ TANYAQKFQG, wherein X ¹ Is V, X ² Is P, and X ³ Is H; and (c) CDR-H3 comprising 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 to the amino acid sequence of SEQ ID NO 75, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO. 61; and (f) CDR-L3 comprising amino acid sequence HQYIHLHSFT of SEQ ID NO: 62; and is

ii) the second antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID No. 90, wherein the VH domain comprises: (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, wherein X is D; and (c) CDR-H3 comprising the amino acid sequence GVGFFXH of SEQ ID NO. 78, wherein 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 to the amino acid sequence of SEQ ID No. 93, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence KAX of SEQ ID NO:79 ¹ EDIYNRX ² A, wherein, X ¹ Is F and X ² Is R; (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, wherein X is W;

or

B) i) the first antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO 71, wherein the VH domain comprises: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO 57, wherein X is I; (b) CDR-H2 comprising the amino acid sequence GX of SEQ ID NO:58 ¹ IX ² IFX ³ TANYAQKFQG, wherein X ¹ Is V, X ² Is P, and X ³ Is H; and (c) CDR-H3 comprising 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 to the amino acid sequence of SEQ ID NO 75, wherein the VL domain comprises: (d) CDR-L1 comprising amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 61; and (f) CDR-L3 comprising amino acid sequence HQYIHLHSFT of SEQ ID NO: 62; and is

ii) the second antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO 91, wherein the VH domain comprises: (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, wherein X is D; and (c) CDR-H3 comprising the amino acid sequence GVGFFXH of SEQ ID NO. 78, wherein X is E;

or

C) i) the first antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO 71, wherein the VH domain comprises: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO 57, wherein X is I or T; (b) CDR-H2, which Amino acid sequence GX comprising SEQ ID NO 58 ¹ IX ² IFX ³ TANYAQKFQG, wherein X ¹ Is V, I or H, X ² Is P or H, and X ³ Is H or G; and (c) CDR-H3 comprising amino acid sequence YDAHYGELDF of SEQ ID NO: 59;

ii) the second antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID No. 90, wherein the VH domain comprises: (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, wherein X is D or E; and (c) CDR-H3 comprising the amino acid sequence GVGFFXH of SEQ ID NO. 78, wherein 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 to the amino acid sequence of SEQ ID No. 94, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence KAX of SEQ ID NO:79 ¹ EDIYNRX ² A, wherein, X ¹ Is F or T and X ² Is R or L; (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, wherein X is W or R;

or

D) i) the first antigen binding site comprises

VH domain sequence having at least 90%, 91%, 92% amino acid sequence of SEQ ID NO 7293%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, wherein the VH domain comprises: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO 57, wherein X is I or T; (b) CDR-H2 comprising the amino acid sequence GX of SEQ ID NO:58 ¹ IX ² IFX ³ TANYAQKFQG, wherein X ¹ Is V, I or H, X ² Is P or H, and X ³ Is H or G; and (c) CDR-H3 comprising amino acid sequence YDAHYGELDF of SEQ ID NO: 59;

ii) the second antigen binding site comprises

or

E) i) the first antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO. 73, wherein the VH domain comprises: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO 57, wherein X is I or T; (b) CDR-H2 comprising the amino acid sequence GX of SEQ ID NO:58 ¹ IX ² IFX ³ TANYAQKFQG, wherein X ¹ Is V, I or H, X ² Is P or H, and X ³ Is H or G; and (c) CDR-H3 comprising amino acid sequence YDAHYGELDF of SEQ ID NO: 59;

ii) the second antigen binding site comprises

And a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID No. 93, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence KAX of SEQ ID NO:79 ¹ EDIYNRX ² A, wherein, X ¹ Is F or T and X ² Is R or L; (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, wherein X is W or R;

or

F) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

and a VL domain sequence having at least 90%, 91%, 92%, 93% amino acid sequence as set forth in SEQ ID NO 94,94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence KAX of SEQ ID NO:79 ¹ EDIYNRX ² A, wherein, X ¹ Is F or T and X ² Is R or L; (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, wherein X is W or R;

or

G) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:92, wherein the VH domain comprises: (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, wherein X is D or E; and (c) CDR-H3 comprising the amino acid sequence GVGFFXH of SEQ ID NO. 78, wherein X is D or E;

or

H) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO 91, wherein the VH domain comprises: (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, wherein X is D or E; and (c) CDR-H3 comprising the amino acid sequence GVGFFXH of SEQ ID NO. 78, wherein X is D or E;

or

I) i) the first antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID No. 72, wherein the VH domain comprises: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO 57, wherein X is I or T; (b) CDR-H2 comprising the amino acid sequence GX of SEQ ID NO:58 ¹ IX ² IFX ³ TANYAQKFQG, wherein X ¹ Is V, I or H, X ² Is P or H, and X ³ Is H or G; and (c) CDR-H3 comprising amino acid sequence YDAHYGELDF of SEQ ID NO: 59;

ii) the second antigen binding site comprises

or

J) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

K) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

L) i) the first antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:74, wherein the VH domain comprises: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO 57, wherein X is I or T; (b) CDR-H2 comprising the amino acid sequence GX of SEQ ID NO:58 ¹ IX ² IFX ³ TANYAQKFQG, wherein X ¹ Is a VI or H, X ² Is P or H, and X ³ Is H or G; and (c) CDR-H3 comprising amino acid sequence YDAHYGELDF of SEQ ID NO: 59;

ii) the second antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID No. 90, wherein the VH domain comprises: (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, wherein X is D or E; and (c) CDR-H3 comprising the amino acid sequence GVGFFXH of SEQ ID NO. 78, wherein X is D or E;

or

M) i) said first antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:74, wherein the VH domain comprises: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO. 57, wherein X is I or T; (b) CDR-H2 comprising the amino acid sequence GX of SEQ ID NO:58 ¹ IX ² IFX ³ TANYAQKFQG, wherein X ¹ Is V, I or H, X ² Is P or H, and X ³ Is H or G; and (c) CDR-H3 comprising amino acid sequence YDAHYGELDF of SEQ ID NO: 59;

ii) the second antigen binding site comprises

or

N) i) the first antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:74, wherein the VH domain comprises: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO 57, wherein X is I or T; (b) CDR-H2 comprising the amino acid sequence GX of SEQ ID NO:58 ¹ IX ² IFX ³ TANYAQKFQG, wherein X ¹ Is V, I or H, X ² Is P or H, and X ³ Is H or G; and (c) CDR-H3 comprising amino acid sequence YDAHYGELDF of SEQ ID NO: 59;

ii) the second antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:92, wherein the VH domain comprises: (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, wherein X is D or E; and (c) CDR-H3 comprising the amino acid sequence GVGFFXH of SEQ ID NO. 78, wherein 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 to the amino acid sequence of SEQ ID No. 93, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence KAX of SEQ ID NO:79 ¹ EDIYNRX ² A, wherein, X ¹ Is F or T and X ² Is R or L; (e) CDR-L2 comprising the amino acid sequence GATSLEH of SEQ ID NO. 80; and (f) a CDR-L3,it comprises the amino acid sequence QQFXSAPYT of SEQ ID NO. 81, wherein X is W or R;

or

O) i) said first antigen binding site comprises

ii) the second antigen binding site comprises

And a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID No. 93, wherein the VL domain comprises: (d) the CDR-L1 is provided with a structure,comprising the amino acid sequence KAX of SEQ ID NO. 79 ¹ EDIYNRX ² A, wherein, X ¹ Is F or T and X ² Is R or L; (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, wherein X is W or R;

or

P) i) said first antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO 71, wherein the VH domain comprises: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO 57, wherein X is I or T; (b) CDR-H2 comprising the amino acid sequence GX of SEQ ID NO:58 ¹ IX ² IFX ³ TANYAQKFQG, wherein X ¹ Is V, I or H, X ² Is P or H, and X ³ Is H or G; and (c) CDR-H3 comprising amino acid sequence YDAHYGELDF of SEQ ID NO: 59;

ii) the second antigen binding site comprises

and a VL junctionA domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO 93, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence KAX of SEQ ID NO:79 ¹ EDIYNRX ² A, wherein, X ¹ Is F or T and X ² Is R or L; (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, wherein X is W or R.

In one embodiment, the bispecific antibodies described herein

i) Blocking the binding of CCL2 to its receptor CCR2 in vitro (reporter gene assay, IC) ₅₀ 0.5 nM); and/or

ii) inhibition of CCL 2-mediated chemotaxis (IC) of myeloid cells in vitro ₅₀ 1.5 nM); and/or

iii) cross-react with cynomolgus monkey CCL2 and human CCL 2.

In one embodiment, the bispecific antibodies described herein do not cross-react with other CCL homologs, particularly show 100-fold reduced binding to other CCL homologs (e.g., CCL8) compared to binding to CCL 2.

In one embodiment, a bispecific antibody described herein binds to a first and a second epitope on human CCL2 in an ion-dependent manner.

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

In one embodiment, the bispecific antibody described herein binds to human CCL2 with 10-fold greater affinity at pH 7.4 than at pH 5.8.

In one embodiment, the bispecific antibody described herein comprises an IgG1 heavy chain constant domain (or Fc domain thereof) 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 to prolong plasma half-life); and/or

iv) Q438R and/or S440E (suitable for inhibiting rheumatoid factor binding).

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 for FcRn to prolong plasma half-life); and/or

iv) Q438R and/or S440E (suitable for inhibiting rheumatoid factor binding).

In one embodiment, the bispecific antibody described herein comprises an IgG1 heavy chain constant domain (or Fc domain thereof) comprising the following mutations (Kabat EU numbering)

i) Q311R and P343R (adapted to increase 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 fcgrs); and

iii) N434A (suitable for increasing affinity for FcRn to prolong plasma half-life); and

iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

i) Q311R and P343R (adapted to increase pI to enhance antigen uptake); and

ii) N434A (suitable for increasing affinity for FcRn to prolong plasma half-life); and

iii) Q438R and/or S440E (useful for inhibiting rheumatoid factor binding).

Q311R and P343R (adapted to increase pI to enhance antigen uptake).

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

iv) Q438R and/or S440E (suitable for inhibiting rheumatoid factor binding).

i) Q311R and P343R (adapted to increase 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 to prolong plasma half-life); and

iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

i) Q311R and P343R (adapted to increase pI to enhance antigen uptake); and

iii) N434A and (adapted to increase affinity for FcRn to prolong plasma half-life); and

iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

In one embodiment, the bispecific antibodies described herein comprise two IgG1 heavy chain constant domains (or Fc domains thereof) comprising (independently or in addition to the mutations described above) the following mutations (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

One embodiment of the invention is an (isolated) (monospecific) antibody that binds (specifically) to human CCL2,

wherein the antibody comprises

A) A VH domain comprising: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO 57, wherein X is I or T; (b) CDR-H2 comprising the amino acid sequence GX of SEQ ID NO:58 ¹ IX ² IFX ³ TANYAQKFQG, wherein X ¹ Is V, I or H, X ² Is P or H, and X ³ Is H or G; and (c) CDR-H3 comprising amino acid sequence YDAHYGELDF of SEQ ID NO: 59;

and

Or alternatively

B) A VH domain 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, wherein X is D or E; and (c) CDR-H3 comprising the amino acid sequence GVGFFXH of SEQ ID NO. 78, wherein X is D or E;

and

wherein the antibody comprises

A) A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75;

or

B) 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;

or

C) A VH domain comprising the amino acid sequence of SEQ ID NO 73;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75;

Or alternatively

D) 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;

or

E) 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

F) 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

G) 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

H) 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 94;

one embodiment of the invention is an isolated nucleic acid encoding a (monospecific or bispecific) antibody according to any one of the preceding embodiments.

One embodiment of the invention is a host cell comprising such a nucleic acid.

One embodiment of the invention is a method of producing an antibody comprising culturing the host cell to produce the antibody.

In one embodiment of the invention, such methods further comprise the step of recovering the antibody from the host cell.

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

One embodiment of the invention is a bispecific antibody as described herein for use as a medicament.

One embodiment of the invention is the use of a bispecific antibody as described herein in the manufacture of a medicament.

In one embodiment, such medicaments are used to treat cancer.

In one embodiment, such medicaments are used to treat an inflammatory or autoimmune disease.

One embodiment of the invention is a bispecific antibody as described herein for use in the treatment of cancer.

One embodiment of the invention is a bispecific antibody as described herein for use in the treatment of an inflammatory or autoimmune disease.

One embodiment of the invention is a method of treating a subject suffering from cancer comprising administering to the subject an effective amount of an antibody described herein.

One embodiment of the invention is a method of treating an individual having an inflammatory or autoimmune disease comprising administering to the individual an effective amount of an antibody described herein.

Drawings

FIG. 1: surface plasmon resonanceThe sensorgram shows that monospecific anti-CCL 2 antibody (CNTO888(═ CNTO), 1a5, 1G9, and humanized 11K2(═ 11K2) bind to recombinant CCL2 and CCL2 homologues.

FIG. 2 a: i.v. serum concentration of hCCL2 over time after injection of preformed immune complexes, including a) solid line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg monospecific anti-CCL 2 antibody CNTO888-SG1 (wild-type IgG1), or b) dotted line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg of the monospecific anti-CCL 2 antibody CNTO888-SG105(Fc receptor binding silent IgG 1).

FIG. 2 b: i.v. serum concentration of hCCL2 over time after injection of preformed immune complexes, including a) solid line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg monospecific anti-CCL 2 antibody 11K2-SG1 (wild-type IgG1), or b) dotted line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg monospecific anti-CCL 2 antibody 11K2-SG105(Fc receptor binding silent IgG 1).

FIG. 2 c: i.v. serum concentration of hCCL2 over time after injection of preformed immune complexes, including a) solid line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg monospecific anti-CCL 2 antibody ABN912-SG1 (wild-type IgG1), or b) dotted line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg monospecific anti-CCL 2 antibody ABN912-SG105(Fc receptor binding silent IgG 1).

FIG. 2 d: i.v. serum concentration of hCCL2 over time after injection of preformed immune complexes, including a) solid line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg monospecific anti-CCL 2 antibody 1a4-SG1 (wild-type IgG1), or b) dotted line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg monospecific anti-CCL 2 antibody 1A4-SG105(Fc receptor binding silent IgG 1).

FIG. 2 e: i.v. serum concentration of hCCL2 over time after injection of preformed immune complexes, including a) solid line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg monospecific anti-CCL 2 antibody 1a5-SG1 (wild-type IgG1), or b) dotted line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg monospecific anti-CCL 2 antibody 1A5-SG105(Fc receptor binding silent IgG 1).

FIG. 2 f: i.v. serum concentration of hCCL2 over time after injection of preformed immune complexes, including a) solid line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg monospecific anti-CCL 2 antibody 1G9-SG1 (wild-type IgG1), or b) dotted line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg monospecific anti-CCL 2 antibody 1G9-SG105(Fc receptor binding silent IgG 1).

FIG. 2 g: i.v. serum concentration of hCCL2 over time after injection of preformed immune complexes, including a) solid line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg monospecific anti-CCL 2 antibody 2F6-SG1 (wild-type IgG1), or b) dotted line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg monospecific anti-CCL 2 antibody 2F6-SG105(Fc receptor binding silent IgG 1).

FIG. 3: time course showing serum total mouse CCL2 concentration (fig. 3a) and antibody-time curve (fig. 3b) after i.v. injection of: 20mg/kg monospecific anti-CCL 2 antibody 11K2-SG1 (wild-type IgG1), and b) dotted line: 20mg/kg monospecific anti-CCL 2 antibody 11K2-SG105(Fc receptor binding silent IgG 1).

FIG. 4 a: i.v. serum concentration of hCCL2 over time after injection of preformed immune complexes, including a) solid line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg bispecific anti-CCL 2 antibody 11K2//1G9-WT IgG1 (wild-type IgG1 binding to the intact Fc receptor), or b) dotted line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg bispecific anti-CCL 2 antibody 11K2//1G9-PGLALA (Fc receptor binding silencing IgG 1).

FIG. 4 b: i.v. serum concentration of hCCL2 over time after injection of preformed immune complexes, including a) solid line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg bispecific anti-CCL 2 antibody CNTO888//11K2-WT IgG1 (wild-type IgG1 binding to intact Fc receptor), or b) dotted line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg bispecific anti-CCL 2 antibody CNTO888//11K2-PGLALA (Fc receptor binding silent IgG 1).

FIG. 4 c: i.v. serum concentration of hCCL2 over time after injection of preformed immune complexes, including a) solid line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg bispecific anti-CCL 2 antibody CNTO888//1G9-WT IgG1 (wild-type IgG1 binding to intact Fc receptor), or b) dotted line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg bispecific anti-CCL 2 antibody 11K2//1G9-PGLALA (Fc receptor binding silencing IgG 1).

FIG. 4 d: i.v. serum concentration of hCCL2 over time after injection of preformed immune complexes, including a) solid line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg bispecific anti-CCL 2 antibody CNTO888//1a5-WT IgG1 (wild-type IgG1 binding to intact Fc receptor), or b) dotted line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg bispecific anti-CCL 2 antibody CNTO888//1A5-PGLALA (Fc receptor binding silent IgG 1).

FIG. 4 e: i.v. serum concentration of hCCL2 over time after injection of preformed immune complexes, including a) solid line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg bispecific anti-CCL 2 antibody 1a5//1G9-WT IgG1 (wild-type IgG1 binding to the intact Fc receptor), or b) dotted line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg bispecific anti-CCL 2 antibody 1A5//1G9-PGLALA (Fc receptor binding silencing IgG 1).

FIG. 4 f: i.v. serum concentration of hCCL2 over time after injection of preformed immune complexes, including a) solid line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg bispecific anti-CCL 2 antibody 11K2//2F6-WT IgG1 (wild-type IgG1 binding to the intact Fc receptor), or b) dotted line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg bispecific anti-CCL 2 antibody 11K2//2F6-PGLALA (Fc receptor binding silencing IgG 1).

FIG. 4 g: i.v. serum concentration of hCCL2 over time after injection of preformed immune complexes, including a) solid line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg bispecific anti-CCL 2 antibody ABN912//11K2-WT IgG1 (wild-type IgG1 binding to the intact Fc receptor), or b) dotted line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg bispecific anti-CCL 2 antibody ABN912//11K2-PGLALA (Fc receptor binding silencing IgG 1).

FIG. 4 h: i.v. serum concentration of hCCL2 over time after injection of preformed immune complexes, including a) solid line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg bispecific anti-CCL 2 antibody 1a4//2F6-WT IgG1 (wild-type IgG1 binding to the intact Fc receptor), or b) dotted line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg bispecific anti-CCL 2 antibody 1A4//2F6-PGLALA (Fc receptor binding silencing IgG 1).

FIG. 4 i: i.v. serum concentration of hCCL2 over time after injection of preformed immune complexes, including a) solid line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg bispecific anti-CCL 2 antibody 1a5//2F6-WT IgG1 (wild-type IgG1 binding to the intact Fc receptor), or b) dotted line: 0.1mg/kg human CCL2(hCCL2) and 20mg/kg bispecific anti-CCL 2 antibody 1A5//2F6-PGLALA (Fc receptor binding silencing IgG 1).

FIG. 5 a:the sensor plots show the binding curves of the four modified 11K2 and four CNTO888 variants at ph7.4 (black line) and ph5.8 (grey line) to monomeric CCL2, and the 16 bispecific anti-CCL 2 antibodies CKLO 01-CKLO 16 produced the results for the four modified 11K2 and four CNTO888 variants each binding to an antigen-binding moiety.

FIG. 5 b:the sensorgram shows the binding curves of monomeric CCL2 for the four modified 11K2 and the four CNTO888 variants, and the results of the 16 bispecific anti-CCL 2 antibodies CKLO01 through CKLO16 producing the four modified 11K2 and the four CNTO888 variants each binding to an antigen-binding moiety. Immediately after the dissociation phase at pH7.4, another dissociation phase at pH5.8 was integratedIn the analysis.

FIG. 6:the sensorgram shows the binding curves of bispecific anti-CCL 2 antibodies CKLO01, CKLO02, CKLO03, and CKLO04 to monomeric CCL8 at ph7.4 (black line) and ph5.8 (grey line).

FIG. 7 a: hCCL2 serum concentrations of a pre-formed immune complex consisting of hCCL2 and bispecific anti-CCL 2 antibodies (parent CNTO//11K2 and pH-dependent variants CKLO01, CKLO02, CKLO03, and CKLO04) were injected into SCID mice for a period of time.

FIG. 7 b: hCCL2 serum concentrations were measured during the latter period of injection of pre-formed immune complexes consisting of hCCL2 and CKLO03 (with IgG1 wild-type Fc) or CKLO03-SG1099 (CKLO 03 with enhanced I Fc) into SCID mice.

FIG. 8: chemotaxis assay: bispecific anti-CCL 2 antibodies with identical CDRs and variable regions VH/VL, i.e., CKLO2-IgG1 wild-type and CKLO2-SG1095, but with different Fc portions, can have the same potency (IC) ₅₀ 0.2 μ g/ml; fig. 8, left panel) inhibits THP-1 cell migration.

Likewise, CCL2-0048 (the parent unmodified bispecific antibody CNTO888/11k2k2 IgG1 of CKLO 2) is pH independent, which also shows IC ₅₀ 0.2. mu.g/ml, since pH dependence is crucial for antigen clearance, this phenomenon does not occur in this assay.

The corresponding monospecific antibody CNTO888 IgG1 and humanized 11k2 IgG1 showed IC ₅₀ Values were 0.3 and 0.7. mu.g/ml, respectively, whereas the huIgG1 isotype control showed no inhibition (FIG. 8, right panel).

FIG. 9: in vivo antitumor activity in a genetically modified mouse model. Treatment of mouse tumor models with Mab CKLO2-IgG1(Fc wild-type IgG1) and CKLO2-SG1099((═ CKLO2pI enhanced) — at the end of the study, tumor volume (left), tumor weight (middle), and M-MDSC infiltration (right) ((vehicle for black bars, CKLO2 wild-type IgG1 for gray bars, and CKLO2 pI-enhanced Fc (CKLO2-SG1099) for white bars/dashed lines))

FIG. 10: during in vivo anti-tumor activity studies, (see efficacy in figure 9) serum total (left) and free (right) CCL2 levels were treated with bispecific anti-CCL 2 antibodies (vehicle for black bars, CKLO2 wild-type IgG1 for gray bars and pI-enhanced Fc (CKLO2-SG1099) for white bars/dashed lines).

FIG. 11: proof of concept study of CCL2 clearance efficiency in cynomolgus macaques. A total antibody concentration-time curve in cynomolgus monkey serum; left panel: the average concentration-time curves for the four antibodies over 7 days are presented; group 1: monospecific CNTO 888-SG 1(═ IgG1 wild-type) anti-CCL 2 antibody (n ═ 3 animals) as a control for maximal total CCL2 accumulation; group 2: biparatopic (biparatopic) anti-CCL 2 antibody CKLO2-SG1(IgG1 wild-type) (n ═ 3) with pH-dependent target binding but without Fc modification; group 3: a biparatopic anti-CCL 2 antibody CKLO2-SG1100 (n-4) with pH-dependent target binding and Fc-pI and further modified; and group 4: a biparatopic anti-CCL 2 antibody CKLO2-SG1095 (n-4) with pH-dependent target binding, enhanced Fc-pI and Fc γ RIIb affinities and further modifications; right panel: individual concentration-time curves for individual 4 (group 2) during the PK study (70 days) are presented.

FIG. 12: proof of concept study of CCL2 clearance efficiency in cynomolgus macaques. Concentration-time curve of total CCL2 in cynomolgus monkey serum; left panel: the average total CCL2 concentration-time curve for the four antibodies over 7 days is presented; right panel: individual total CCL2 concentration-time curves for individual 4 (group 2) during the PK study (70 days) are presented.

FIG. 13: concentration-time curve of free CCL2 in cynomolgus monkey serum; left panel: the mean free CCL2 concentration-time curve for the four antibodies over 7 days is presented; right panel: individual free CCL2 concentration-time curves for individual 4 (group 2) during the PK study (70 days) are presented; for samples below the detection limit, an average curve was calculated using a value of 0.01ng/mL (lower limit of quantitation).

FIG. 14: PK/PD study of CCL2 clearance efficiency in cynomolgus macaques. Total CKL02-SG1095 concentration-time curve in cynomolgus monkey serum (CKL 02-SG1095 treatment at different concentrations (groups 1-3)); left panel: the average concentration-time curve for the three dose levels over 7 days (n-4) is presented; right panel: individual concentration-time curves for two individual ADA-negative animals (25mg/kg dose group) over the study period (98 days) are presented.

FIG. 15: PK/PD study of CCL2 clearance efficiency in cynomolgus macaques. Total CCL2 concentration-time profiles in cynomolgus monkey serum at different concentrations of CKL02-SG1095 (groups 1-3) and compared to CNTO888-SG1 (group 4); left panel: the mean total CCL2 concentration-time curve (error bars represent Sd) for the four study groups over 7 days is presented; right panel: individual total CCL2 concentration-time curves are presented for ADA-negative animals in group 3 (n-2, error bars indicate range) and group 4 (n-3, error bars indicate SD) during the PK study (day 98).

FIG. 16: PK/PD study of CCL2 clearance efficiency in cynomolgus macaques. Concentration-time curve of free CCL2 in cynomolgus monkey serum; left panel: mean free CCL2 concentration-time curves (error bars indicate SD) for the four study groups over 7 days were presented (CKL 02-SG1095 treatment at different concentrations (groups 1-3) and comparison with CNTO888-SG1 treatment (group 4); right panel: mean free CCL2 concentration-time curves from ADA-negative animals of group 3 (n-2, error bars indicate range) and group 4 (n-3, error bars indicate SD) during the PK study (day 70) are presented; for samples below the detection limit, an average curve was calculated using a value of 0.01ng/mL (lower limit of quantitation).

Detailed Description

The present invention relates to bispecific anti-CCL 2 antibodies that bind to two different epitopes on human CCL2, pharmaceutical compositions thereof, their manufacture, and use as medicaments for the treatment of cancer, inflammatory diseases, autoimmune diseases, and ophthalmic diseases. Thus, the antibody comprises a first antigen binding site that (specifically) binds to a first epitope on human CC2 and a second, different antigen binding site that (specifically) binds to a second, different epitope.

The invention includes bispecific antibodies 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,

Wherein the content of the first and second substances,

In one embodiment, when a preformed immune complex consisting of 20mg/kg of each bispecific antibody and 0.1mg/kg of human CCL2 is administered to an FcRn transgenic mouse at a single dose of 10ml/kg, the in vivo clearance (ml/day/kg) of human CCL2 after administration of the bispecific antibody comprising an fcgamma receptor silencing constant heavy chain domain of the human IgG1 isotype (or the Fc domain thereof) comprising the mutations L234A, L235A, P329G (Kabat EU numbering) is at least 15-fold, particularly at least 20-fold higher than the in vivo clearance (ml/day/kg) of human CCL2 after administration of the bispecific antibody comprising a constant heavy chain domain of the human IgG1 isotype (or the Fc domain thereof) of human wild-type IgG 1.

The term "epitope" includes any polypeptide determinant capable of specific binding to an antibody. In certain embodiments, epitope determinants include chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural characteristics and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. The present invention relates to bispecific anti-CCL 2 antibodies that bind to two different epitopes on human CCL 2. The terms first and second epitope relate to two different epitopes on human CCL 2. Thus, the second epitope is different from the first epitope, and it can be readily determined whether an antibody binds to the same epitope as, or competes for binding with, the reference anti-CCL 2 antigen-binding site by using conventional methods known in the art. For example, to determine whether a test antibody binds to the same epitope as the reference anti-CCL 2 antigen binding site of the invention, a reference antibody is allowed to bind to the CCL2 domain under saturating conditions. Second, the ability of the test antibody to bind human CCL2 was evaluated. If the test antibody is capable of binding to human CCL2 after saturation binding to the reference anti-CCL 2 antigen binding site, it can be concluded that the test antibody binds to a different epitope than the reference anti-CCL 2 antigen binding site. On the other hand, if the test antibody is unable to bind human CCL2 after saturation binding to the reference anti-CCL 2 antigen binding site, the test antibody can bind to the same epitope as the reference anti-CCL 2 antibody of the invention. Additional routine experimentation (e.g., peptide mutation and binding analysis) can then be performed to confirm that the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody or that steric blockade (or other phenomena) is responsible for the unobserved binding. This classification can be done using ELISA, RIA, surface plasmon resonance (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 invention, two antibodies are considered to bind to the same (or overlapping) epitope if one antibody inhibits binding of the other antibody by at least 50%, at least 75%, at least 90%, or even 99% in a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess as measured by a competitive binding assay (see, e.g., Junghans et al, Cancer Res.1990:50: 1495-.

Alternatively, two antibodies are considered to bind the same epitope if substantially all of the amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody. Two antibodies are considered to have an "overlapping epitope" if the amino acid mutation set that one antibody binds is reduced or eliminated, while the binding of the other antibody is reduced or eliminated.

To determine whether the antibody competes for binding with the reference anti-CCL 2 antibody, the binding method described above was performed in two orientations: in a first orientation, a reference antibody was allowed to bind CCL2 under saturation conditions, and then the binding of the test antibody to human CCL2 was assessed. In the second orientation, the test antibody was allowed to bind to CCL2 molecules under saturating conditions, and then the binding of the reference antibody to human CCL2 was assessed. If in this orientation only the first (saturating) antibody is able to bind to the CCL2 molecule, it can be concluded that the test antibody and the reference antibody compete for binding to CCL 2. As will be understood by those having ordinary skill in the art, an antibody that competes for binding with a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding to an overlapping or adjacent epitope.

As used herein, the terms "CCL 2", "human CCL 2" (also referred to as "MCP-1") mean the 76 amino acid sequence referenced in NCBI record accession No. NP _002973, and are referred to as CCL2, MCP-1 (monocyte chemotactic protein 1), SMC-CF (smooth muscle cell chemokine), LDCF (lymphocyte-derived chemokine), GDCF (glioma-derived monocyte chemokine), TDCF (tumor-derived chemokine), HCl1 (human cytokine 11), MCAF (monocyte chemokine and activator), respectively. The gene symbol is SCYA2, JE gene on chromosome 17 of human, and the new name is CCL2(ZLotnik, Yoshie 2000.Immunity 12: 121-Asan 127). JE is a mouse homolog of human MCP-1/CCL 2.

Hendrel (Handel) et al (biochemistry. 1996; 35: 6569-6584) determined the solution structure of the CCL2 dimer. These studies indicate that the secondary structure of CCL2 consists of four β -sheets. Furthermore, Zhang and Rollins (Mol Cell biol. 1995; 15: 4851-4855) describe the residues responsible for the dimerization interface of CCL 2. The protein complex appears to be very elongated and the two monomers are oriented such that they form a large pocket. The structure of the two crystal forms of monomeric and dimeric CCL2, the so-called I and P forms, was also determined (Lubkowski et al, Nat Struct biol. 1997; 4: 64-69). (Paolini et al J Immunol.1994Sep 15; 153(6):2704-17) describe the presence of MCP1/CCL2 as monomers at physiologically relevant concentrations: the function of CCL2 protein (purchased from Peprotech) by particle size screening HPLC, sedimentation equilibrium ultracentrifugation and chemical cross-linking was recorded by analysis, which might show that the weight fraction of MCP-1 monomeric and dimeric forms depends on the in vitro concentration. Finally, Seo and coworkers (J Am Chem Soc.2013Mar 20; 135(11):4325-32) might show ion mobility mass spectrometry, with injected CCL2 present as monomers and dimers under physiological conditions.

Thus, "wild-type CCL-2" (wt CCL2) may be present as a monomer, but in practice dimers may also be formed at physiological concentrations. This monomer-dimer equilibrium is of course different and must be carefully considered in all in vitro experiments where different concentrations may be used. To avoid any uncertainty, we generated point mutation CCL2 variants: the "P8A" variant of CCL2 carries mutations in the dimerization interface, resulting in the inability to form dimers that result in a defined pure CCL2 monomer. In contrast, the "T10C" variant of CCL2 resulted in an immobilized dimer of CCL2 (J Am Chem Soc.2013Mar 20; 135 (11): 4325-32).

The CCL2/CCR2 axis is the primary mediator that recruits immature myeloid cells into tumors. CCL2 is overexpressed by malignant cells and binds to the extracellular matrix (ECM), establishing a chemotactic gradient. Once they reach the tumor, myeloid-derived suppressor cells (MDSCs) in turn suppress the initiation of anti-tumor T cell responses by secreting/upregulating anti-inflammatory cytokines/receptors, contributing to the original tumor environment. In this way, MDSCs can reduce or even impair the efficacy of any T cell activation therapy (Meyer et al, 2014). Thus, specific inhibition of recruitment of these immature myeloid cells would improve the efficacy of checkpoint inhibitors, T cell bispecific and cancer immunotherapy. In addition, CCL2 is also associated with promoting angiogenesis, metastasis, and tumor growth, suggesting that neutralization of CCL2 may contribute to a variety of anti-tumor interventions.

In contrast to its receptor, targeting CCL2 will specifically inhibit the undesired CCL 2-mediated effects, thereby avoiding those signals that might be emitted by the same receptor (CCR2) 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.

CCL2 is a preferred antibody target in some studies clinically directed to neutralizing elevated levels caused by different 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, their high synthesis rate and observed high in vivo antibody-antigen dissociation constant (KD) have been demonstrated to be major obstacles preventing conventional antibodies from inhibiting free CCL2 at clinically feasible doses (Fetterly et al, 2013).

Neutralization of CCL2 appears to be more significantly associated with patients with elevated serum CCL2 levels, which has been observed in various types of cancer, such as Breast Cancer (BC), ovarian cancer (OvCa), colorectal cancer (CRC), pancreatic cancer, and prostate cancer. However, even though patients within these indications did not exhibit such serology but had tumors that were highly infiltrated by myeloid immune cells, patients may benefit from this novel treatment due to the multiple roles CCL2 plays in the tumor environment as described above.

As used herein, an antibody "binds to human CCL2," "specifically binds to human CCL2," "binds to human CCL2," or "anti-CCL 2 antibody" relates to binding with K _D Value of 5.0x10 ^-8 An antibody that specifically binds human CCL2 antigen with a binding affinity of mol/l or less, in one embodiment, K _D Value of 1.0x10 ^-9 mol/l or less, in one embodiment, K _D The value was 5.0x10 ^-8 mol/l to 1.0x10 ^-13 mol/l。

Binding affinity is determined by standard binding assays, e.g. surface plasmon resonance techniques (CGE-Healthcare Uppsala, Sweden), for example using a construct comprising the extracellular domain of CCL2 (e.g. its naturally occurring 3-dimensional structure). In one embodiment, binding affinity was determined in a standard binding assay using exemplary soluble CCL 2.

Antibody specificity involves the selective recognition of an antibody against a particular epitope of an antigen. For example, natural antibodies are monospecific.

As used herein, the term "monospecific" antibody refers to an antibody having one or more binding sites, each binding site binding to the same epitope of the same antigen.

As used herein, the terms "biparatopic antibody that binds to (human) CCL2," "bispecific antibody that binds to (human) CCL2," "biparatopic anti-CCL 2 antibody," "bispecific anti-CCL 2 antibody" mean that the antibody is capable of specifically binding to at least two different epitopes on (human) CCL 2. Generally, such bispecific antibodies comprise two different antigen binding sites (two different paratopes), each specific for a different epitope of (human) CCL 2. In certain embodiments, the bispecific antibody is capable of binding two different and non-overlapping epitopes on CCL2, meaning that two different antigen binding sites do not compete for binding to CCL 2.

An "acceptor human framework" is for purposes herein a framework derived from the amino acid sequence of a human immunoglobulin framework or human consensus framework, comprising a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework, as defined below. The acceptor human framework that is "derived from" a human immunoglobulin framework or human consensus framework may comprise the same amino acid sequence as it, or it may contain variations in the amino acid sequence. In some embodiments, the number of amino acid changes 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 identical to a sequence of a VL human immunoglobulin framework sequence or a human consensus framework sequence.

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

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

As used herein, the term "valency" means the presence of a specified number of antigen binding sites in an antibody. Thus, the term "monovalent binding to an antigen" means that there is one (and no more than one) antigen binding site in the antibody that is specific for the antigen.

The term "antigen binding site" relates to a site or region, i.e. one or more amino acid residues, that provides an antibody that interacts with an antigen. For example, the antigen binding site of an antibody comprises amino acid residues from a Complementarity Determining Region (CDR). In one embodiment, the antigen binding site of the antibody comprises amino acid residues from VH and VL. Natural immunoglobulin molecules typically have two antigen binding sites, and Fab molecules typically have a single antigen binding site. An "antigen-binding portion" relates to a polypeptide molecule comprising an antigen-binding site that specifically binds to an antigenic determinant. Antigen binding portions include antibodies and fragments thereof as further defined herein. Particular antigen-binding portions include the antigen-binding domain of an antibody, which comprises an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, the antigen-binding portion can include an antibody constant region as further defined herein and known in the art. Useful heavy chain constant regions include any of the five isoforms, α, δ, ε, γ or μ. Useful light chain constant regions include any of two isoforms: κ and λ.

As used herein, the term "antigenic determinant" or "antigen" refers to a site on a polypeptide macromolecule that binds to an antigen-binding moiety/site to form an antigen-binding moiety-antigen complex. For example, useful antigenic determinants may be present 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, absent from serum, and/or present in the extracellular matrix (ECM).

The term "chimeric" antibody relates to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The "class" of an antibody refers to the constant region or type of constant region that its heavy chain has. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of them may be further divided into subclasses (isotypes), e.g., IgG ₁ 、IgG ₂ 、IgG ₃ 、IgG ₄ 、IgA ₁ And IgA ₂ . The heavy chain constant regions corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. Preferably, the bispecific antibody of the invention is of human IgG isotype, more preferably of human IgG1 isotype. The terms IgG isotype and IgG1 isotype as used herein relate to the human IgG isotype and the human IgG1 isotype. In general, different IgG isotypes exist in slightly different allotypic forms based on allelic variation between IgG subclasses (see Vidarsson et al; Front Immunol 5(2014) Article 520, 1-17). An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective to achieve a desired therapeutic or prophylactic effect at a desired dosage and for a desired period of time.

The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain may vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from Cys226 or Pro230 to the carboxy-terminus of the heavy chain. However, the antibody produced by the host cell may undergo post-translational cleavage of one or more, in particular one or two, amino acids at the C-terminus of the heavy chain. Thus, an antibody produced by a host cell by expression of a particular nucleic acid molecule encoding a full-length heavy chain may comprise the full-length heavy chain, or may comprise a cleaved variant of the full-length heavy chain (also referred to herein as a "cleaved variant heavy chain"). The last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbered according to the Kabat EU index). Thus, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (K447) of the Fc region may or may not be present. Unless otherwise indicated, the amino acid sequence of the heavy chain comprising the Fc domain (or a subunit of the Fc domain as defined herein) is referred to herein as being free of the C-terminal glycine-lysine dipeptide. In one embodiment of the invention, the heavy chain (subunit comprising an Fc domain as specified herein) comprised by the antibody or bispecific antibody according to the invention comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In one embodiment of the invention, the heavy chain (subunit comprising an Fc domain as specified herein) comprised by the antibody or bispecific antibody according to the invention comprises an additional C-terminal glycine residue (G446, numbering according to the Kabat EU index). A composition of the invention, e.g., a pharmaceutical composition described herein, comprising an antibody or population of bispecific antibodies of the invention. The population of antibodies or bispecific antibodies can comprise molecules having full-length heavy chains and molecules having cleaved variant heavy chains. The population of antibodies or bispecific antibodies can consist of a mixture of molecules having full length heavy chains and molecules having cleavage variant heavy chains, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the antibodies or bispecific antigen binding molecules have cleavage variant heavy chains. In one embodiment of the invention, a composition comprising a population of antibodies or bispecific antibodies of the invention comprises an antibody or bispecific antibody comprising a heavy chain comprising subunits of an Fc domain as specified herein and an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In one embodiment of the invention, a composition comprising a population of antibodies or bispecific antibodies of the invention comprises an antibody or population of bispecific antibodies comprising a heavy chain comprising a subunit of an Fc domain as specified herein and an additional C-terminal glycine residue (G446, numbering according to the Kabat EU index). In one embodiment of the invention, such a composition comprises a population of antibodies or bispecific antibodies consisting of: a molecule comprising a heavy chain comprising a subunit of an Fc domain as specified herein; a molecule comprising a heavy chain comprising a subunit of an Fc domain as specified herein and an additional C-terminal glycine residue (G446, numbered according to the Kabat EU index); and molecules comprising subunits comprising an Fc domain as specified herein and an additional heavy chain of C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is "according to the EU numbering system," also referred to as "EU index numbering according to Kabat" or "Kabat EU numbering," as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD,1991 (supra). As used herein, a "subunit" of an Fc domain refers to one of two polypeptides that form a dimeric Fc domain, i.e., a polypeptide comprising a C-terminal constant region of an immunoglobulin heavy chain that is capable of stably self-association. For example, subunits of the IgG Fc domain comprise IgG CH2 and IgG CH3 constant domains.

"framework" or "FR" refers to variable domain residues other than the hypervariable region (HVR) residues. The FRs of a variable domain typically consist of four FR domains: FR1, FR2, FR3 and FR 4. Thus, HVR and FR sequences typically occur in the VH (or VL) in the following order: FR-H1(L1) -CDR-H1(L1) -FR-H2(L2) -CDR-H2(L2) -FR-H3(L3) -CDR-H3(L3) -FR-H4 (L4).

The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region as defined herein.

A "human antibody (human antibody)" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source using the human antibody repertoire (antibody repertoire) or other human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues.

A "human consensus framework" is a framework that represents the most common amino acid residues in a series of human immunoglobulin VL or VH framework sequences. Typically, the series of human immunoglobulin VL or VH sequences is derived from a subset of variable domain sequences. In general, the subgroups of Sequences are those as in Kabat, E.A. et al, Sequences of Proteins of Immunological Interest, 5 th edition, Bethesda MD (1991), NIH Publication 91-3242, Vols.1-3. In one embodiment, for VL, the subgroup is subgroup kappa I as described in Kabat et al, supra. In one embodiment, for the VH, the subgroup is subgroup III as described in Kabat et al, supra.

"humanized" antibodies refer to chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody can optionally comprise at least a portion of an antibody constant region derived from a human antibody. "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

As used herein, the term "complementarity determining region" or "CDR" refers to each region of an antibody variable domain that is hypervariable in sequence and/or forms structurally defined loops ("hypervariable loops") and/or comprises antigen-contacting residues ("antigen contacts"). Generally, an antibody comprises six HVRs; three in VH (CDR-H1, CDR-H2, CDR-H3) and three in VL (CDR-L1, CDR-L2, CDR-L3). Herein, exemplary CDRs include:

(A) hypervariable loops are present 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-;

(B) CDRs are present 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, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991));

(C) antigen contacts were present 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) (iii) a combination of (a), (b) and/or (c) comprising 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).

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

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

An "isolated" antibody is one that is separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, 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 assessing antibody purity, see, e.g., Flatman, s. et al, j.chromatogr.b 848(2007) 79-87.

An "isolated" nucleic acid is a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally includes the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from the natural chromosomal location.

An "isolated nucleic acid encoding one or more bispecific anti-CCL 2 antibodies" relates to one or more nucleic acid molecules encoding the heavy and light chains of an antibody (or fragments thereof), including such nucleic acid molecules in a single vector or in isolated vectors, and such nucleic acid molecules being present at one or more locations in a host cell.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., antibodies that are identical and/or bind to the same epitope(s) of a subject antibody contained in the population, but does not include, for example, antibodies that contain naturally occurring mutations or possible variants resulting from the production of monoclonal antibody preparations, such variants typically being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates that the characteristics of the antibody are obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be made by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals comprising all or part of a human immunoglobulin locus, which methods and other exemplary methods for making monoclonal antibodies are described herein.

"Natural antibodies" relates to naturally occurring immunoglobulin molecules having different structures. For example, a natural IgG antibody is a heterotetrameric glycoprotein composed of two identical light chains and two identical heavy chains that are disulfide-bonded at about 150,000 daltons. From N-terminus to C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH 3). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), also known as a variable light domain or light chain variable domain, followed by a light chain Constant (CL) domain. The light chain of an antibody can be divided into two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant region.

The term "package insert" is used to refer to instructions typically included in commercial packaging for a therapeutic product that include information regarding the indications, usage, dosage, route of administration, combination therapy, contraindications, and/or warnings with which such therapeutic product is used.

"percent (%) amino acid residue identity," with respect to a reference polypeptide sequence, refers to the percentage of amino acid residues in a candidate sequence that are the same as the amino acid residues in the participating polypeptide sequence, and after aligning the sequences and introducing differences, if necessary, the maximum percentage of sequence identity can be achieved, and without regard to any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein, the use of the sequence comparison computer program ALIGN-2 results in% amino acid sequence identity values. The ALIGN-2 sequence comparison computer program was written by GeneTake, Inc., and the source code has been deposited with the user files in the U.S. copyright office, Washington, D.C., 20559, and registered with U.S. copyright registration number TXU 510087. The ALIGN-2 program is publicly available from GeneTak corporation, san Francisco, Calif., or may be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system (including digital UNIX V4.0D). All sequence comparison parameters were set by the ALIGN-2 program and were not changed.

In the case of amino acid sequence comparisons using ALIGN-2, the% amino acid sequence identity of a given amino acid sequence a to or relative to a given amino acid sequence B (which may alternatively be expressed as given amino acid sequence a having or comprising a certain% amino acid sequence identity to or relative to a given amino acid sequence B) is calculated as follows:

100 times a fraction X/Y

Wherein X is the number of amino acid residues in the alignment of programs A and B that are scored as identical matches by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B. It will be understood that where 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 will not be 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 using the ALIGN-2 computer program as described in the preceding paragraph.

The term "pharmaceutical formulation" refers to a formulation in a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain other components that have unacceptable toxicity to the subject to which the formulation will be administered.

By "pharmaceutically acceptable carrier" is meant an ingredient of a pharmaceutical formulation other than an active ingredient that is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

As used herein, "treatment" (and grammatical variants thereof, such as "treatment" or "treating") refers to a clinical intervention that attempts to alter the natural course of a disease in a subject being treated, and may be prophylactic or performed during the course of clinical pathology. Desirable therapeutic effects include, but are not limited to, preventing occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating a disease state, alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the progression of the disease or slow the progression of the disease.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) are typically of similar structure and each domain comprises four conserved Framework Regions (FR) and three hypervariable regions (HVRs). (see, e.g., Kindt, t.j. et al, Kuby Immunology, 6 th edition, w.h.freeman and co., n.y. (2007), page 91). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind a particular antigen can be isolated from antibodies that bind the antigen using VH or VL domains to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano, S. et al, J.Immunol.150(1993) 880-; clackson, T.et al, Nature 352(1991) 624-.

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors which are self-replicating nucleic acid structures as well as vectors which are incorporated into the genome of a host cell. Certain vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".

I. Compositions and methods

In one aspect, the invention is based in part on the following findings: the bispecific antibodies described herein use different anti-CCL 2 antigen binding sites as the first and second antigen binding sites/moieties. These anti-CCL 2 antibodies bind certain epitopes of CCL2 with high specificity and have the ability to specifically inhibit the binding of CCL2 to its receptor CCR 2. They showed improved immune complex formation and improved elimination of CCL2 in vivo compared to monospecific antibodies.

Bispecific anti-CCL 2 antibodies

Bispecific antibodies

The bispecific antibodies described herein are monoclonal antibodies having binding specificity for at least two different epitopes on CCL 2.

Techniques for making multispecific and bispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs of different specificity (see Milstein and Cuello, Nature 305:537(1983)) and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168, and Atwell et al, J.mol.biol.270:26 (1997)). Multispecific antibodies can also be prepared by: engineered electrostatic steering effects for the preparation of antibody Fc-heterodimer molecules (see, e.g., WO 2009/089004); crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980; and Brennan et al, Science,229:81 (1985)); use leucones zippers to product bi-specific antibodies (see, e.g., Kostelny et al, J.Immunol.,148(5):1547-1553(1992) and WO 2011/034605); the problem of light chain mismatches is circumvented using the commonly used light chain technology (see e.g. WO 98/50431); bispecific antibody fragments were prepared using the "diabody" technique (see, e.g., Hollinger et al, Proc. Natl. Acad. Sci. USA,90: 6444-; and the use of single chain fv (sFv) dimers (see, e.g., Gruber et al, J.Immunol.,152:5368 (1994)); and the preparation of trispecific antibodies, such as those described in Tutt et al, J.Immunol.147:60 (1991).

Also included herein are engineered antibodies having three or more antigen binding sites, including, for example, "octopus antibodies" or DVD-Ig (see, e.g., WO 2001/77342 and WO 2008/024715). Further examples of multispecific antibodies having three or more antigen binding sites may be found in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO 2010/145792 and WO 2013/026831. Bispecific antibodies or antigen-binding fragments thereof also include "dual-acting fabs" or "DAFs" comprising an antigen-binding site that binds to CCL2 as well as to another different antigen or to two different epitopes of CCL2 (see, e.g., US 2008/0069820 and WO 2015/095539).

Multispecific antibodies may also be provided in an asymmetric form comprising domains that cross in one or more binding arms with the same antigen specificity, i.e. by exchanging VH/VL domains (see e.g. WO 2009/080252 and WO 2015/150447), CH1/CL domains (see e.g. WO 2009/080253) or the complete Fab arm (see e.g. WO 2009/080251, WO 2016/016299, see also Schaefer et al, PNAS, 108(2011)1187-1191, and Klein et al, MAbs 8(2016)1010-20), also known as CrossMabs. Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations into the domain interface to direct proper Fab pairing. See, for example, WO 2016/172485.

Various other molecular forms for multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al, Mol Immunol 67(2015) 95-106).

Preferred bispecific antibody formats.

According to particular embodiments of the invention, bispecific antibodies described herein comprise domains that cross in one or more binding arms with the same antigen specificity, i.e. by exchanging VH/VL domains (see e.g. WO 2009/080252 and WO 2015/150447), CH1/CL domains (see e.g. WO 2009/080253) or the complete Fab arm (see e.g. WO 2009/080251, WO 2016/016299, see also Schaefer et al, PNAS,108(2011)1187-1191, and Klein at, MAbs 8(2016) 1010-20).

Charge modification in such bispecific antibodies, especially those with VH/VL domain exchange (see WO 2015/150447): the bispecific antibodies of the present invention may comprise therein an amino acid substitution in the Fab molecule comprised which is particularly effective in reducing light chain mismatches with non-matching heavy chains (Bence-Jones type by-products) which may occur in the preparation of Fab-based bi/multispecific antigen-binding molecules in which a VH/VL exchange occurs in one of its binding arms (or more if the molecule comprises more than two antigen-binding Fab molecules) (see also PCT publication No. WO 2015/150447, particularly the examples therein, which are incorporated herein by reference in their entirety). The ratio of desired bispecific antibody to undesired by-products, particularly the Bence Jones type by-products occurring in bispecific antibodies with VH/VL domain exchange in one of their binding arms, can be improved by introducing charged amino acids with opposite charges at specific amino acid positions in the CH1 and CL domains (sometimes referred to herein as "charge modification").

Thus, in some embodiments, wherein the first and second antigen-binding portions of the bispecific antibody are both Fab molecules, and in one of the antigen-binding portions (in particular the second antigen-binding portion), the variable domains VL and VH of the Fab light chain and Fab heavy chain are substituted for each other,

i) in the constant domain CL of the first antigen-binding portion, the amino acid at position 124 is substituted with a positively charged amino acid (numbering according to Kabat), and wherein, in the constant domain CH1 of the first antigen-binding portion, the amino acid at position 147 or the amino acid at position 213 is substituted with a negatively charged amino acid (numbering according to Kabat EU index); or

ii) in the constant domain CL of the second antigen-binding portion the amino acid at position 124 is substituted with a positively charged amino acid (numbering according to Kabat), and wherein in the constant domain CH1 of the second antigen-binding portion the amino acid at position 147 or the amino acid at position 213 is substituted with a negatively charged amino acid (numbering according to Kabat EU index).

The bispecific antibody does not comprise the modifications described under i) and ii). The constant domains CL and CH1 of the antigen-binding portion with the VH/VL exchange were not swapped with each other (i.e. left in an un-swapped state).

In a more particular embodiment of the method of the present invention,

i) in the constant domain CL of the first antigen-binding portion, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbering according to Kabat), and in the constant domain CH1 of the first antigen-binding portion, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index); or

ii) in the constant domain CL of the second antigen-binding portion the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CH1 of the second antigen-binding portion the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

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

In another embodiment, in the constant domain CL of the first antigen-binding portion the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbering according to Kabat), and in the constant domain CH1 of the first antigen-binding portion the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

In a particular embodiment, in the constant domain CL of the first antigen-binding portion the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CH1 of the first antigen-binding portion the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

In a more particular embodiment, in the constant domain CL of the first antigen-binding portion the amino acid at position 124 is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) (numbering according to Kabat), and in the constant domain CH1 of the first antigen-binding portion the amino acid at position 147 is substituted with glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to Kabat EU index).

In an even more particular embodiment, in the constant domain CL of the first antigen-binding portion the amino acid at position 124 is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with arginine (R) (numbering according to Kabat), and in the constant domain CH1 of the first antigen-binding portion the amino acid at position 147 is substituted with glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to Kabat EU index).

In a specific embodiment, the constant domain CL of the first antigen-binding portion is of the kappa isotype if the amino acid substitutions according to the above embodiments occur in the constant domain CL and in the constant domain CH1 of the first antigen-binding portion.

Alternatively, amino acid substitutions according to the above embodiments may occur in the constant domain CL and constant domain CH1 of the second antigen-binding portion, rather than in the constant domain CL and constant domain CH1 of the first antigen-binding portion. In particular, in this embodiment, the constant domain CL of the second antigen-binding portion is of the kappa isotype.

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

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

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

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

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

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

In another embodiment according to these aspects of the invention, in the first subunit of the Fc domain the serine residue at position 354 is in turn substituted with a cysteine residue (S354C) or the glutamic acid residue at position 356 is substituted with a cysteine residue (E356C) (in particular the serine residue at position 354 is substituted with a cysteine residue), and in the second subunit of the Fc domain the tyrosine residue at position 349 is in turn substituted with a cysteine residue (Y349C) (numbering according to Kabat EU index).

Alternative heterodimerization techniques are described below in the context of "Fc domains" and are also considered to be other embodiments of the invention.

In another embodiment according to these aspects of the invention, the Fc domain is human IgG ₁ An Fc domain.

Fc domains and modifications

In particular embodiments, bispecific antibodies of the invention comprise an Fc domain comprised of a first subunit and a second subunit. It will be appreciated that the features described herein with respect to the Fc domain of a bispecific antibody are equally applicable to the Fc domain constituted in an antibody of the invention.

The Fc domain of a bispecific antibody consists of a pair of polypeptide chains comprising the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin g (IgG) molecule is a dimer, each subunit of which comprises a CH2 and CH3 IgG heavy chain constant domain. The two subunits of the Fc domain are capable of stably binding to each other. In one embodiment, the bispecific antibody of the invention comprises no more than one Fc domain.

In one embodiment, the Fc domain of the bispecific antibody is an IgG Fc domain. In a particular embodiment, the Fc domain is IgG ₁ An Fc domain. In another embodiment, the Fc domain is IgG ₄ An Fc domain. In a more specific embodiment, the Fc domain is IgG ₄ An Fc domain comprising an amino acid substitution at position S228 (numbered according to the Kabat EU index), in particular the amino acid substitution S228P. This amino acid substitution reduces IgG in vivo ₄ Fab arm exchange of antibodies (see Stubenrauch et al, Drug Metabolism and Disposition 38, 84-91 (2010)). In another particular embodiment, the Fc domain is a human Fc domain. In an even more particular embodiment, the Fc domain is a human IgG ₁ An Fc domain.

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

Fc domain modification to promote heterodimerization

Bispecific antibodies according to the invention comprise different antigen-binding portions, which may be fused to one or the other of the two subunits of the Fc domain, whereby the two subunits of the Fc domain are typically comprised in two different polypeptide chains. Recombinant co-expression and subsequent dimerization of these polypeptides results in several possible combinations of the two polypeptides. To improve the yield and purity of bispecific antibodies in recombinant production, it would be advantageous to introduce modifications in the Fc domain of bispecific antibodies that facilitate the desired polypeptide association.

Thus, in particular embodiments, the Fc domain of a bispecific antibody according to the invention comprises a modification that facilitates association of the first and second subunits of the Fc domain. The most extensive protein-protein interaction site between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one embodiment, the modification is in the CH3 domain of the Fc domain.

There are a number of methods for modifying the CH3 domain of the Fc domain in order to enhance heterodimerization, which are well described in e.g. WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, in all of these approaches, the CH3 domain of the first unit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are both engineered in a complementary manner such that each CH3 domain (or heavy chain comprising a CH3 domain) is no longer able to homodimerize with itself, but is forced to heterodimerize with the other CH3 domain that is complementarily engineered (such that the first CH3 domain and the second CH3 domain heterodimerize and no homodimer is formed between the two first CH3 domains or the two second CH3 domains). These different approaches for improving heavy chain heterodimerization are considered to be different options for binding to heavy chain-light chain modifications in bispecific antibodies (e.g., VH and VL exchange/substitutions in one binding arm, and substitutions that introduce oppositely charged amino acids in the CH1/CL interface), which reduce heavy chain/light chain mismatches and Bence Jones type by-products.

In one particular embodiment, the modification that facilitates 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.

The "protrusion-into-hole" technique is described, for example, in: US 5,731,168; US 7,695,936; ridgway et al, Prot Eng 9, 617. sup. 621 (1996); and Carter, J immunological Meth248, 7-15 (2001). Generally, the method comprises introducing a protrusion ("protuberance") at the interface of the first polypeptide and a corresponding cavity ("well") in the interface of the second polypeptide such that the protrusion can be positioned in the cavity, thereby promoting heterodimer formation and hindering homodimer formation. The protuberance is constructed by replacing a smaller amino acid side chain on the first polypeptide interface with a larger side chain (e.g., tyrosine or tryptophan). By replacing the larger amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine), a complementary cavity of the same or similar size as the protuberance is formed in the interface of the second polypeptide.

Thus, in particular embodiments, in the CH3 domain of the first subunit of the Fc domain of the bispecific antibody, an amino acid residue is substituted with an amino acid residue having a larger side chain volume, thereby creating a protuberance within the CH3 domain of the first subunit that is positionable in a cavity within 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 substituted with an amino acid residue having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit within which a protuberance within the CH3 domain of the second subunit is positionable.

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

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

The projections and cavities can be made by altering the nucleic acid encoding the polypeptide, for example by mutagenesis at specific sites or by peptide synthesis.

In one particular embodiment, in the (CH3 domain) of the first subunit of the Fc domain (the "bulge" subunit), the threonine residue at position 366 is substituted with a tryptophan residue (T366W), and in the (CH3 domain) of the second subunit of the Fc domain (the "pore" subunit), the tyrosine residue at position 407 is substituted with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, the threonine residue at position 366 is in turn substituted with a serine residue (T366S), and the leucine residue at position 368 is substituted with an alanine residue (L368A) (numbering according to the Kabat EU index).

In yet another embodiment, in the first subunit of the Fc domain, the serine residue at position 354 is in turn substituted with a cysteine residue (S354C) or the glutamic acid residue at position 356 is substituted with a cysteine residue (E356C) (in particular the serine residue at position 354 is substituted with a cysteine residue), and in the second subunit of the Fc domain, the tyrosine residue at position 349 is in turn substituted with a cysteine residue (Y349C) (numbering according to the Kabat EU index). The introduction of these two cysteine residues results in the formation of disulfide bonds between the two subunits of the Fc domain, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

In one particular embodiment, the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering according to the Kabat EU index).

In particular embodiments, the antigen binding portion that binds to the second antigen is fused to the first subunit of the Fc domain (comprising a "knob" modification) (optionally, by binding to CCL2 and/or a peptide linker). Without wishing to be bound by theory, fusion of an antigen-binding moiety that binds to a second antigen (e.g., an activated T cell antigen) to the knob-containing subunit of the Fc domain will (further) minimize the production of a polypeptide comprising two antigen-binding moieties that bind to an activated T cell antigen (spatial collisions of two knob-containing polypeptides).

Other techniques for CH3 modification for forced heterodimerization can be envisaged as alternatives to the present invention and are described in, for example, WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO 2013/096291.

In one embodiment, the heterodimerization method described in EP 1870459 may alternatively be used. The method is based on the introduction of oppositely charged amino acids at specific amino acid positions of the CH3/CH3 domain interface between the two subunits of the Fc domain. A preferred embodiment of the bispecific antibody of the invention is the amino acid mutations R409D and K370E in one of the two CH3 domains (of the Fc domain); and amino acid mutations D399K and E357K (numbering according to the Kabat EU index) in the other of the two CH3 domains of the Fc domain.

In another embodiment, the bispecific antibody of the invention comprises the amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and the amino acid mutations R409D, K370E in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations D399K, E357K in the CH3 domain of the second subunit of the Fc domain (numbering according to the Kabat EU index).

In another embodiment, the bispecific antibody of the invention comprises the amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain or said bispecific antibody comprises the amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations S354C, T S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain and the amino acid mutations R D, K370E in the CH 82409 domain of the first subunit of the Fc domain and the amino acid mutations D K, E357K in the CH3 domain of the second subunit of the Fc domain (all numbering according to the Kabat index).

In one embodiment, the heterodimerization process described in WO 2013/157953 can be used alternatively. In one embodiment, the first CH3 domain comprises the amino acid mutation T366K and the second CH3 domain comprises the amino acid mutation L351D (numbering according to the EU index of Kabat). In another embodiment, the first CH3 domain further comprises the amino acid mutation L351K. In another embodiment, the second CH3 domain further comprises an amino acid mutation selected from the group consisting of Y349E, Y349D and L368E (preferably L368E) (numbering according to the EU index of Kabat).

In one embodiment, the heterodimerization process described in WO 2012/058768 may alternatively be used. In one embodiment, the first CH3 domain comprises the amino acid mutations L351Y, Y407A, and the second CH3 domain comprises the amino acid mutations T366A, K409F. In another embodiment, the second CH3 domain further comprises an amino acid mutation at position T411, D399, S400, F405, N390, or K392 selected from, for example: a) T411N, T411R, T411Q, T411K, T411D, T411E, or T411W; b) D399R, D399W, D399Y or D399K; 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 (numbering according to the Kabat EU index). In another embodiment, the first CH3 domain comprises the amino acid mutations L351Y, Y407A and the second CH3 domain comprises the amino acid mutations T366V, K409F. In another embodiment, the first CH3 domain comprises the amino acid mutation Y407A and the second CH3 domain comprises the amino acid mutations T366A, K409F. In another embodiment, the second CH3 domain further comprises the amino acid mutations K392E, T411E, D399R and S400R (numbering according to the EU index of Kabat).

In one embodiment, the heterodimerization approach described in WO 2011/143545 may alternatively be used, e.g., with amino acid modifications at positions selected from the group consisting of 368 and 409 (numbering according to the EU index of Kabat).

In one embodiment, the heterodimerization process described in WO 2011/090762 can be used instead, again using the "bulge-in-hole" technique described above. In one embodiment, the first CH3 domain comprises the amino acid mutation T366W and the second CH3 domain comprises the amino acid mutation Y407A. In one embodiment, the first CH3 domain comprises the amino acid mutation T366Y and the second CH3 domain comprises the amino acid mutation Y407T (numbering according to the EU index of Kabat).

In one embodiment, the bispecific antibody or Fc domain thereof is of IgG ₂ Subclass, and alternatively the heterodimerization method described in WO 2010/129304.

In an alternative embodiment, the modification that facilitates association of the first and second subunits of the Fc domain comprises a modification that mediates electrostatic steering, for example as described in PCT publication WO 2009/089004. Typically, this method involves substituting one or more amino acid residues at the interface of two Fc domain subunits with charged amino acid residues such that homodimer formation is electrostatically unfavorable, but heterodimerization is electrostatically favorable. In one such embodiment, the first CH3 domain comprises amino acid substitutions of K392 and N392 of negatively charged amino acids (e.g. glutamic acid (E) or aspartic acid (D), preferably K392D or N392D) and the second CH3 domain comprises amino acid substitutions of positively charged amino acids (e.g. lysine (K) or arginine (R), preferably D399K, E356K, D356K or E357K and more preferably D399K and E356K) to D399, E356, D356 or E357. In another embodiment, the first CH3 domain further comprises a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), more preferably K409D or R409D) substituted for the amino acid of K409 or R409. In another embodiment, the first CH3 domain further or alternatively comprises an amino acid substitution of a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D)) to K439 and/or K370 (all numbered according to the Kabat EU index).

In yet another embodiment, the heterodimerization process described in WO 2007/147901 may alternatively be used. In one embodiment, the first CH3 domain comprises the amino acid mutations K253E, D282K, and K322D, and the second CH3 domain comprises the amino acid mutations D239K, E240K, and K292D (numbering according to the Kabat EU index).

In another embodiment, the heterodimerization process described in WO 2007/110205 can be used instead.

In one embodiment, the first subunit of the Fc domain comprises amino acid substitutions K392D and K409D, and the second subunit of the Fc domain comprises amino acid substitutions D356K and D399K (numbering according to the Kabat EU index).

As used herein, the term "wild-type (WT) IgG or IgG 1" for a bispecific anti-CCL 2 antibody relates to a bispecific antibody comprising an IgG or IgG1 constant heavy chain, which may comprise the above-described modifications/mutations that promote heterodimerization, but which does not comprise further Fc domain modifications/mutations that increase or decrease Fc receptor binding and/or effector function, as described below.

Fc domain modifications/mutations that increase or decrease Fc receptor binding and/or effector function

Modification of bispecific anti-CCL 2 antibodies by scavenging techniques

Bispecific anti-CCL 2 antibodies were modified using a sweeping technique to enable bispecific anti-CCL 2 antibodies to eliminate free CCL2 over a longer period of time, thereby enabling sustained biological effects, such as anti-cancer efficacy, in vivo.

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

The present invention provides a method for promoting antigen uptake into cells mediated by an antibody by reducing the antigen binding activity (binding ability) of the above antibody in an acidic pH range to less than its antigen binding activity in a neutral pH range; this facilitates antigen uptake into the cell. The invention also provides methods of promoting antigen uptake into a cell mediated by an antibody based on altering at least one amino acid in the antigen binding domain of the above antibody to promote antigen uptake into a cell. The present invention also provides methods for promoting antigen uptake into cells mediated by antibodies based on the substitution of at least one amino acid with histidine or the insertion of at least one histidine into the antigen binding domain of the above antibodies to promote antigen uptake into cells.

Herein, "antigen uptake into cells" mediated by antibodies refers to the uptake of antigen into cells by endocytosis. Meanwhile, herein, "promoting uptake into cells" means that the intracellular uptake rate of antibodies bound to antigens in plasma is increased, and/or the amount of the ingested antigens recycled to plasma is decreased. This means that the rate of uptake into cells is promoted compared to the antibody before increasing the human FcRn binding activity of the antibody in the neutral pH range, or compared to the antibody before increasing the human FcRn binding activity in the acidic pH range and reducing the antigen binding activity (binding capacity) of the antibody to less than its antigen binding activity in the neutral pH range. The rate is preferably increased compared to intact human IgG, and more preferably compared to intact human IgG. Thus, in the present invention, it can be assessed whether an antibody promotes antigen uptake into a cell based on an increase in the rate of antigen uptake into the cell. The rate of antigen uptake into a cell can be calculated, for example, by: after adding antigen and antibody to the medium, the decrease in the concentration of antigen in the medium containing human FcRn-expressing cells is monitored over time, or the amount of antigen taken up into human FcRn-expressing cells is monitored over time. The methods of the invention are used to promote the rate of antigen uptake into cells mediated by antibodies, e.g., the rate of antigen elimination from plasma can be increased by administering antibodies. Thus, it can also be assessed whether antibody-mediated antigen uptake into cells is promoted, e.g., by testing whether the rate of antigen removal from plasma is accelerated via administration of the antibody or whether the total antigen concentration in plasma is reduced.

Herein, "total antigen concentration in plasma" refers to the sum of the concentration of antibody-bound antigen and antibody-unbound antigen, or "free antigen concentration in plasma", i.e., the concentration of antibody-unbound antigen. Various methods of measuring "total antigen concentration in plasma" or "free antigen concentration in plasma" are well known in the art, as described below.

As used herein, "whole human IgG" (or "wild-type (WT) human IgG") refers to unmodified human IgG (except for potential modifications with respect to heterodimerization described above) and is not limited to a particular class of IgG. This means that human IgG1, IgG2, IgG3, or IgG4 can be used as "fully human IgG" as long as it can bind to human FcRn in an acidic pH range. Preferably, the "fully human IgG" may be human IgG 1.

The invention also provides methods of increasing the number of antigens to which a single antibody can bind. More specifically, the invention provides methods for increasing the amount of antigen that a single antibody can bind to, the antigen for which human FcRn binding activity is possible in the acidic pH range by increasing the human FcRn binding activity of the antibody in the neutral pH range. The invention also provides a method of increasing the amount of antigen that a single antibody that has human FcRn binding activity at an acidic pH range can bind to by altering at least one amino acid in the human FcRn binding domain of the antibody.

The present invention provides methods for promoting antigen uptake into cells mediated by antibodies. More specifically, the present invention provides methods for promoting antigen uptake into cells by antibodies having human FcRn binding activity in the acidic pH range, based on increasing the human FcRn binding activity of the antibody in the neutral pH range. The invention also provides methods for improving antigen uptake into cells by antibodies having human FcRn binding activity in the acidic pH range based on altering at least one amino acid in the human FcRn binding domain of the antibody.

The invention also provides a method for facilitating antigen uptake into a cell by an antibody having human FcRn binding activity in the acidic pH range based on the use of a human FcRn binding domain comprising an amino acid sequence that replaces with a different amino acid at least one amino acid selected from the following positions in the parent IgG Fc domain of a human FcRn binding domain comprising the Fc domain of a parent IgG: 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).

The present invention also provides a method for promoting antigen uptake into cells mediated by an antibody by reducing the antigen binding activity (binding ability) of the above antibody in an acidic pH range to less than its antigen binding activity in a neutral pH range; this facilitates antigen uptake into the cell. The invention also provides methods of promoting antigen uptake into a cell mediated by an antibody based on altering at least one amino acid in the antigen binding domain of the above antibody to promote antigen uptake into a cell. The present invention also provides methods for promoting antigen uptake into cells mediated by antibodies based on the substitution of at least one amino acid with histidine or the insertion of at least one histidine into the antigen binding domain of the above antibodies to promote antigen uptake into cells.

As used herein, "intact human IgG" (or "wild-type IgG") refers to unmodified human IgG (except for potential modifications with respect to heterodimerization described above) and is not limited to a particular class of IgG. This means that human IgG1, IgG2, IgG3, or IgG4 can be used as "fully human IgG" as long as it can bind to human FcRn in an acidic pH range. Preferably, the "fully human IgG" may be human IgG 1.

As used herein, "parent IgG" refers to unmodified IgG that is subsequently modified to produce a variant, so long as the modified variant of the parent IgG can bind to human FcRn at acidic pH ranges (thus, the parent IgG does not need to have binding activity to human FcRn under acidic conditions). The parent IgG may be a naturally occurring IgG, or a variant or engineered form of a naturally occurring IgG. A parent IgG may refer to the polypeptide itself, the composition comprising the parent polypeptide, or the amino acid sequence encoding the parent polypeptide. It should be noted that "parent IgG" includes known commercial, recombinantly produced IgG as described below. The source of the "parent IgG" is not limited and may be obtained from any organism other than a human animal or human. Preferably, the organism is selected from the group consisting of mouse, rat, guinea pig, hamster, gerbil, cat, rabbit, dog, goat, sheep, cow, horse, camel and non-human primate. In another embodiment, the "parent IgG" may also be obtained from cynomolgus macaques, marmosets, rhesus monkeys, chimpanzees, or humans. Preferably, the "parent IgG" is obtained from human IgG1, but is not limited to a particular class of IgG. This means that human IgG1, IgG2, IgG3 or IgG4 may be suitable for use as "parent IgG". In a similar manner, any class or subclass of IgG from any organism above may preferably be used as a "parent IgG". Examples of variants or engineered forms of naturally occurring IgG are described in Curr Opin biotechnol.2009dec; 20(6) 685-91, Curr Opin Immunol.2008Aug; 460-70 (20) (460-70), Protein Eng Des Sel.2010Apr; 23(4) 195-202, WO 2009/086320, WO 2008/092117, WO 2007/041635 and WO 2006/105338, but are not limited thereto.

The invention also provides methods of increasing the ability to eliminate plasma antigens by administering antibodies. In the present invention, "a method of increasing the ability to eliminate a plasma antigen" is synonymous with "a method of increasing the ability of an antibody to eliminate an antigen from plasma". More specifically, the present invention provides methods for increasing the ability to deplete plasma antigens from antibodies that have human FcRn binding activity in the acidic pH range by increasing the human FcRn binding activity of the antibodies in the neutral pH range. The invention also provides a method for increasing the ability to eliminate plasma antigens by an antibody having human FcRn binding activity in the acidic pH range, based on altering at least one amino acid in the human FcRn binding domain of the antibody.

The present invention also provides a method for increasing the capacity to eliminate plasma antigens by an antibody having human FcRn binding activity in the acidic pH range by using a human FcRn binding domain comprising an amino acid sequence of at least one amino acid substitution selected from the following positions in the parent IgG Fc domain of a human FcRn binding domain comprising an Fc domain of a parent IgG having different amino acids: 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 number).

The present invention also provides a method for increasing the ability to eliminate plasma antigens by an antibody having an improved ability to eliminate plasma antigens as compared with antigen-binding activity in a neutral pH range by decreasing the antigen-binding activity of the above antibody in an acidic pH range. The present invention also provides a method for increasing the ability to eliminate plasma antigens by an antibody by altering at least one amino acid in the antigen binding domain of the above antibody having an improved ability to eliminate plasma antigens. The present invention also provides a method for increasing the ability to eliminate plasma antigens by administering an antibody by substituting at least one amino acid with histidine or inserting at least one histidine into the antigen binding domain of the above-described antibody having an improved ability to eliminate plasma antigens.

Herein, "the ability to eliminate plasma antigens" refers to the ability to eliminate antigens from plasma when antibodies are administered or secreted in vivo. Thus, "increasing the ability of an antibody to eliminate plasma antigens" refers herein to accelerating the rate of antigen elimination from plasma following administration of the antibody, prior to increasing the human FcRn binding activity of the antibody in the neutral pH range, or prior to increasing human FcRn binding activity and simultaneously decreasing its antigen binding activity in the acidic pH range to less than the neutral pH range. The increase in the activity of an antibody to eliminate an antigen from plasma can be assessed, for example, by administering a soluble antigen and an antibody in vivo, and measuring the concentration of the soluble antigen in plasma after administration. When the concentration of soluble antigen in plasma after administration of soluble antigen and antibody is decreased by increasing the human FcRn binding activity of the antibody in the neutral pH range, or by increasing the human FcRn binding activity thereof while decreasing the antigen concentration thereof, the binding activity in the acidic pH range is lower than that in the neutral pH range, and it can be judged that the ability of the antibody to eliminate plasma antigen is enhanced. The soluble antigen may be in the form of antibody-bound antigen or antibody-unbound antigen, and the concentrations thereof may be determined as "antibody-bound antigen concentration in plasma" and "antibody-unbound antigen concentration in plasma" (the latter being synonymous with "free antigen concentration in plasma"), respectively. Since "total antigen concentration in plasma" refers to the sum of the concentrations of antibody-bound antigen and antibody-unbound antigen, or "free antigen concentration in plasma", i.e., the concentration of antibody-unbound antigen, the concentration of soluble antigen can be determined as "total antigen concentration in plasma". As described below, various methods for measuring "total antigen concentration in plasma" or "free antigen concentration in plasma" are well known in the art.

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

The present invention also provides a method of improving the pharmacokinetics of an antibody having human FcRn binding activity in the acidic pH range by using a human FcRn binding domain comprising an amino acid sequence that replaces with a different amino acid at least one amino acid selected from the following positions in a parent IgG Fc domain of a human FcRn binding domain comprising an Fc domain of IgG: 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).

The plasma concentration of unbound antibody free antigen or the ratio of free antigen concentration to total concentration can be determined by methods known to those skilled in the art, e.g., by Pharm res.2006jan; 23(1) 95-103. Alternatively, where an antigen exhibits a particular function in vivo, it can be assessed whether the antigen binds to an antibody that neutralizes the function of the antigen (an antagonist molecule) by testing whether the function of the antigen is neutralized. Whether antigen function is neutralized can be assessed by assaying for in vivo markers that reflect antigen function. Whether an antigen binds to an antibody that activates the function of the antigen (an anti-molecule) can be assessed by assaying for in vivo markers that reflect the function of the antigen.

The 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, the in vivo labeling determination and such measurements are not particularly limited; however, the assay is preferably performed after a certain time has elapsed after administration of the antibody. In the present invention, the period of time after the antibody is administered is not particularly limited, and a person skilled in the art can determine an appropriate time depending on the characteristics of the antibody administered and the like. Such times include, for example, one day after administration of the antibody, three days after administration of the antibody, seven days after administration of the antibody, 14 days after administration of the antibody, and 28 days after administration of the antibody. Herein, "plasma antigen concentration" refers to the "total antigen concentration in plasma" or "free antigen concentration in plasma" of the sum of the antibody-bound antigen and the antibody-unbound antigen concentration, i.e., the antibody-unbound antigen concentration.

By administering 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or even higher-fold of an antibody of the invention compared to administration of a reference antibody comprising an intact human IgG Fc domain as a human FcRn binding domain, or compared to administration without an antigen binding domain molecule of the invention, the total antigen concentration in plasma can be reduced.

In another aspect, the invention provides bispecific anti-CCL 2 antibodies that exhibit pH-dependent binding characteristics. As used herein, the expression "pH-dependent binding" refers to an antibody that exhibits "reduced binding to CCL2 at acidic pH as compared to its binding at neutral pH" (for the purposes of this disclosure, the two expressions are used interchangeably). For example, an antibody that "has pH-dependent binding characteristics" includes an antibody that binds CCL2 with higher affinity at neutral pH than at acidic pH. In certain embodiments, a bispecific antibody of the invention binds to CCL2 at neutral pH with at least 2-fold, 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 200-fold, 400-fold, 1000-fold, 10000-fold, or more affinity than at acidic pH. In some embodiments, the antibody binds CCL2 with higher affinity at pH7.4 than at pH 5.8. In other embodiments, the antibody binds to CCL2 at ph7.4 with at least 2-fold, 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 200-fold, 400-fold, 1000-fold, 10000-fold, or more affinity than at ph 5.8.

When the antigen is a soluble protein, binding of the antibody to the antigen can result in an increase in the half-life of the antigen in plasma (i.e., a decrease in the clearance of the antigen from the plasma), because the antibody may have a longer half-life in plasma than the antigen itself and may act as a carrier for the antigen. This is due to the recycling of antigen-antibody complexes through the intracellular body pathway of cells by FcRn (Roopenian, nat. rev. immunol.7(9):715-725 (2007)). However, since antibodies bind to their antigens in the neutral extracellular environment while releasing them into the acidic endosomal compartment upon entry into the cell, antibodies with pH-dependent binding characteristics have superior characteristics in terms of antigen neutralization and clearance relative to their counterparts bound in a pH-independent manner (Igawa et al, Nature Biotechnol.28(11):1203-1207 (2010); Deviabioyin et al, mAbs 5(6):851-859 (2013); WO 2009/125825).

For the purposes of this disclosure, the "affinity" of an antibody for CCL2 is expressed as the KD of the antibody. The KD of an antibody is related to the equilibrium dissociation constant of antibody-antigen interactions. The greater the KD value of an antibody binding to its antigen, the weaker the binding affinity for a particular antigen. Thus, as used herein, the expression "higher affinity at neutral pH than at acidic pH" (or equivalently the expression "pH-dependent binding") refers to an antibody that binds to CCL2 at acidic pH with a KD that is greater than the KD of an antibody that binds to CCL2 at neutral pH. For example, in the context of the present invention, an antibody that binds to CCL2 at acidic pH has a KD at least 2-fold greater than the KD of an antibody that binds to CCL2 at neutral pH is considered to have a higher affinity for binding to CCL2 at neutral pH than at acidic pH. Thus, the invention includes antibodies that bind to CCL2 at acidic pH with a KD at least 2-fold, 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 200-fold, 400-fold, 1000-fold, 10000-fold or more greater than the KD of an antibody that binds to CCL2 at neutral pH. In another example, the antibody can have a KD value at neutral pH of 10-7M, 10-8M, 10-9M, 10-10M, 10-11M, 10-12M, or less. In another example, the antibody can have a KD value at acidic pH of 10-9M, 10-8M, 10-7M, 10-6M, or greater.

In other embodiments, an antibody that binds to CCL2 at pH5.8 is considered to have a higher affinity at neutral pH than at acidic pH if the KD of the antibody is at least 2-fold greater than the KD of the antibody that binds to CCL2 at pH 7.4. In some embodiments, an antibody provided that binds to CCL2 at ph5.8 has a KD at least 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 200-fold, 400-fold, 1000-fold, 10000-fold, or more greater than the KD of an antibody that binds to CCL2 at ph 7.4. In another example, the antibody can have a KD value at pH7.4 of 10-7M, 10-8M, 10-9M, 10-10M, 10-11M, 10-12M, or less. In another example, the antibody can have a KD value at pH5.8 of 10-9M, 10-8M, 10-7M, 10-6M, or greater.

The binding properties of an antibody to a particular antigen can also be expressed in kd for the antibody. The kd of an antibody relates to the dissociation rate constant of the antibody with respect to a particular antigen and is expressed in reciprocal seconds (i.e., sec-1). An increase in kd indicates that the antibody binds less strongly to its antigen. Thus, the invention includes antibodies that bind to CCL2 at a higher kd value at acidic pH than at neutral pH. The invention includes an antibody that binds CCL2 at an acidic pH, with a kd at least 2-fold, 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 200-fold, 400-fold, 1000-fold, 10000-fold or more greater than the kd of an antibody that binds CCL2 at a neutral pH. In another example, the kd value of an antibody at neutral pH can be 10-2X 1/s, 10-3X 1/s, 10-4X 1/s, 10-5X 1/s, 10-6X 1/s, or less. In another example, the kd value of an antibody at acidic pH can be 10-31/s, 10-21/s, 10-11/s, or greater. The invention also includes antibodies that bind to CCL2 having a higher kd value at ph5.8 than at ph 7.4. The invention includes an antibody that binds to CCL2 at ph5.8 with a kd at least 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 200-fold, 400-fold, 1000-fold, 10000-fold, or more greater than the kd of an antibody that binds to CCL2 at ph 7.4. In another embodiment, the kd value of the antibody may be 10-21/s, 10-31/s, 10-41/s, 10-51/s, 10-61/s or less at pH 7.4. In another embodiment, the kd value of the antibody may be 10-31/s, 10-21/s, 10-11/s or greater at pH 5.8.

In certain instances, "reduced binding to CCL2 at acidic pH compared to binding at neutral pH" is expressed as the ratio of the KD value of an antibody binding to CCL2 at acidic pH to the KD value of an antibody binding to CCL2 at neutral pH (and vice versa). For example, for purposes of the present invention, an antibody may be considered to "have reduced binding to CCL2 at acidic pH as compared to its binding at neutral pH" if the antibody exhibits an acidic/neutral KD ratio of 2 or greater. In certain embodiments, an anti-CCL 2 antibody of the invention has a ph5.8/ph7.4KD ratio of 2 or greater. In certain exemplary embodiments, the acidic/neutral KD ratio of an antibody of the invention can be 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 greater. In another example, the antibody can have a KD value at neutral pH of 10-7M, 10-8M, 10-9M, 10-10M, 10-11M, 10-12M, or less. In another example, the antibody can have a KD value at acidic pH of 10-9M, 10-8M, 10-7M, 10-6M, or greater. In other cases, an antibody may be considered to "bind less to CCL2 at acidic pH than to its binding at neutral pH" if the antibody exhibits a pH5.8/pH7.4kd ratio of 2 or greater. In certain exemplary embodiments, the antibody can have a ph5.8/ph7.4KD ratio of 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 greater. In another example, the antibody can have a KD value at pH7.4 of 10-7M, 10-8M, 10-9M, 10-10M, 10-11M, 10-12M, or less. In another example, the antibody can have a KD value at pH5.8 of 10-9M, 10-8M, 10-7M, 10-6M, or greater.

In certain instances, "reduced binding to CCL2 at acidic pH compared to binding at neutral pH" is expressed as the ratio of the kd value of an antibody binding to CCL2 at acidic pH to the kd value of an antibody binding to CCL2 at neutral pH (and vice versa). For example, for purposes of the present invention, an antibody can be considered to "have reduced binding to CCL2 at acidic pH as compared to its binding at neutral pH" if the antibody exhibits an acidic/neutral kd ratio of 2 or greater. In certain exemplary embodiments, the antibodies of the invention have a pH5.8/pH7.4 kd ratio of 2 or greater. In certain exemplary embodiments, the acidic/neutral kd ratio of an antibody of the invention can be 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 greater. In another example, the kd value of an antibody at neutral pH can be 10-2X 1/s, 10-3X 1/s, 10-4X 1/s, 10-5X 1/s, 10-6X 1/s, or less. In another example, the kd value of an antibody at acidic pH can be 10-31/s, 10-21/s, 10-11/s, or greater. In certain exemplary embodiments, the antibody of the invention can have a ph5.8/ph7.4 kd ratio of 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 greater. In another embodiment, the kd value of the antibody may be 10-21/s, 10-31/s, 10-41/s, 10-51/s, 10-61/s or less at pH 7.4. In another embodiment, the kd value of the antibody may be 10-31/s, 10-21/s, 10-11/s or greater at pH 5.8.

As used herein, the expression "acidic pH" refers to a pH of 4.0 to 6.5. The expression "acidic pH" includes any pH value of 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 a particular aspect, the "acidic pH" is 5.8.

As used herein, the expression "neutral pH" refers to a pH of 6.7 to about 10.0. The expression "neutral pH" includes any pH value of 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 a particular aspect, the "neutral pH" is 7.4.

As expressed herein, KD values and KD values can be determined using surface plasmon resonance-based biosensors to characterize antibody-antigen interactions. KD and KD values can be determined at 25 degrees celsius or 37 degrees celsius.

In another aspect, the invention provides bispecific anti-CCL 2 antibodies that form an immune complex (i.e., an antigen-antibody complex) with CCL 2. In certain embodiments, two or more bispecific anti-CCL 2 antibodies bind two or more CCL2 molecules to form an immune complex. This is possible because CCL2 exists as a homodimer containing two CCL2 molecules, whereas antibodies have two antigen binding sites.

In general, when two or more antibodies form immune complexes with two or more antigens, the resulting immune complexes can strongly bind to Fc receptors present on the cell surface due to affinity forces by the Fc region of the antibodies in the complexes, and then can be efficiently taken into cells. Thus, an anti-CCL 2 antibody described above, capable of forming an immune complex comprising two or more anti-CCL 2 antibodies and two or more CCL2 molecules, can bind strongly to Fc receptors due to affinity forces, resulting in rapid clearance of CCL2 from plasma in vivo.

Furthermore, antibodies with pH-dependent binding characteristics are considered to have superior characteristics with respect to their counterparts that bind in a pH-independent manner with respect to antigen neutralization and clearance (Igawa et al, Nature Biotech.28(11):1203-1207 (2010); Deviabioyin et al, mAbs5(6):851-859 (2013); WO 2009/125825). Therefore, an antibody having the above-described two characteristics, i.e., an antibody having a pH-dependent binding characteristic and forming an immune complex comprising two or more antibodies having two or more antigens, is expected to have a more excellent characteristic to eliminate the antigens from plasma at a high speed (WO 2013/081143).

In one aspect, the invention provides a polypeptide comprising a variant Fc region having enhanced fcyriib binding activity, comprising at least two amino acid alterations, comprising: (a) an amino acid change at position 236, and (b) at least one amino acid change 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, according to EU numbering.

In one aspect, the invention provides a polypeptide comprising a variant Fc region having enhanced Fc γ RIIb binding activity, comprising an amino acid change at position 236 according to EU numbering.

In one aspect, the invention provides a polypeptide comprising a variant Fc region having enhanced fcyriib binding activity, comprising at least two amino acid changes, comprising: (a) an amino acid change at position 236, and (b) at least one amino acid change at least one position selected from the group consisting of: 231. 232, 235, 239, 268, 295, 298, 326, 330 and 396, according to EU numbering. In another embodiment, the variant Fc region comprises an amino acid change at least one position selected from the group consisting of: 231. 232, 235, 239, 268, 295, 298, 326, 330 and 396, according to EU numbering. In another embodiment, the variant Fc region comprises an amino acid change at least one position selected from the group consisting of: 268. 295, 326 and 330, according to EU numbering.

In another aspect, the present invention provides a polypeptide comprising a variant Fc region having enhanced fcyriib binding activity, comprising an amino acid change in any one of the following (1) to (37): (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) positions 235, 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, 326 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) positions 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, according to EU numbering.

In another embodiment, the variant Fc region having enhanced fcyriib 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 at position 231; (b) ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 232; (c) asp at position 233; (d) trp, Tyr at position 234; (e) trp at position 235; (f) ala, Asp, Glu, His, Ile, Leu, Met, Asn, Gln, Ser, Thr, Val at position 236; (g) asp, Tyr at position 237; (h) glu, Ile, Met, Gln, Tyr at position 238; (i) ile, Leu, Asn, Pro, Val at position 239; (j) ile at position 264; (k) phe at position 266; (l) Ala, His, Leu at position 267; (m) Asp, Glu at position 268; (n) Asp, Glu, Gly at position 271; (o) Leu at position 295; (p) Leu at position 298; (q) Glu, Phe, Ile, Leu at position 325; (r) a Thr at position 326; (s) Ile, Asn at position 327; (t) Thr at position 328; (u) a Lys, Arg at position 330; (v) glu at position 331; (w) Asp at position 332; (x) Asp, Ile, Met, Val, Tyr at position 334; and (y) Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 396; according to EU numbering.

In another embodiment, the variant Fc region having enhanced fcyriib binding activity comprises at least one amino acid alteration (e.g., substitution) selected from the group consisting of: (a) gly, Thr at position 231; (b) ASP at 232-bit; (c) trp at position 235; (d) asn, Thr at position 236; (e) val at position 239; (f) asp, Glu at position 268; (g) leu not at 295; (h) leu at position 298; (i) thr at position 326; (j) lys, Arg at position 330; (k) lys, Met at position 396; according to EU numbering. In another embodiment, a variant Fc region having enhanced fcyriib binding activity comprises the following amino acid alterations (e.g., substitutions): an Asn at position 236, a Glu at position 268, a Lys at position 330, a Met at position 396; according to EU numbering. In another embodiment, a variant Fc region having enhanced fcyriib binding activity comprises the following amino acid alterations (e.g., substitutions): asn at position 236, Asp at position 268, Lys at position 330; according to EU numbering. In another embodiment, a variant Fc region having enhanced fcyriib binding activity comprises the following amino acid alterations (e.g., substitutions): asn at position 236, Asp at position 268, Leu at position 295, Lys at position 330; according to EU numbering. In another embodiment, a variant Fc region having enhanced fcyriib binding activity comprises the following amino acid alterations (e.g., substitutions): thr at position 236, Asp at position 268, Lys at position 330; according to EU numbering. In another embodiment, a variant Fc region having enhanced fcyriib binding activity comprises the following amino acid alterations (e.g., substitutions): asn at position 236, Asp at position 268, Leu at position 295, Thr at position 326, Lys at position 330; according to EU numbering. In another embodiment, a variant Fc region having enhanced fcyriib binding activity comprises the following amino acid alterations (e.g., substitutions): trp at position 235, Asn at position 236, Asp at position 268, Leu at position 295, Thr at position 326, Lys at position 330; according to EU numbering.

In another aspect, the present invention provides an isolated polypeptide comprising a variant Fc region having an increased isoelectric point (pI). In certain embodiments, the variant Fc region described herein comprises at least two amino acid alterations in the parent Fc region. In certain embodiments, each amino acid change increases the isoelectric point (pI) of the variant Fc region as compared to the isoelectric point of the parent Fc region. They are based on the following findings: for example, when an antibody is administered into the body, an antibody that increases the pI by modifying at least two amino acid residues can promote clearance of an antigen in plasma.

In the present invention, the pI may be a theoretically or experimentally determined pI. The value of pI can be determined, for example, by isoelectric focusing as known to those skilled in the art. Theoretical pI values can be calculated using, for example, gene and amino acid sequence analysis software (Genetyx, etc.).

In one embodiment, the pI value may be increased, e.g., by 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, as compared to prior to modification.

In certain embodiments, the amino acids for increased pI may be exposed on the surface of the variant Fc region. In the present invention, amino acids that can be exposed on the surface generally relate to amino acid residues located on the surface of the polypeptide constituting the variant Fc region. Amino acid residues located on the surface of polypeptides relate to amino acid residues whose side chains can be brought into contact with solvent molecules, usually predominantly water molecules. However, the side chain does not necessarily have to be in complete contact with the solvent molecule, and even if a part of the side chain is in contact with the solvent molecule, the amino acid is defined as "surface-located amino acid residue". Amino acid residues located on the surface of the polypeptide also include amino acid residues located close to the surface and thus may be affected by the charge of another amino acid residue, the side chain of which is even partially in contact with the solvent molecule. One skilled in the art can, for example, use commercially available software to prepare homology models for polypeptides. Alternatively, methods known to those skilled in the art, such as X-ray crystallography, may be used. For example, using a calculator program such as the insight II program (Accelrys), the coordinates from the three-dimensional model are used to determine amino acid residues that can be exposed on the surface. The sites that the surface can expose 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)) software suitable for protein modeling and three-dimensional structural information can be used to determine sites that can be exposed to surfaces. For example, SYBYL Biopolymer Module software (Tripos Associates.) when the algorithm requires a user to enter a size parameter, the "size" of the probe used in the calculation can be set to a radius of about 1.4 angstroms or less furthermore Pacios has described a method of determining the surface exposable area using personal computer software (comput. chem.18(4):377-386 (1994); model.1:46-53 (1995). based on the information as described above, appropriate amino acid residues located on the surface of the polypeptide constituting the variant Fc region can be selected.

In certain embodiments, the polypeptide comprises 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 a biological fluid (e.g., plasma, interstitial fluid, lymphatic fluid, ascites, and pleural fluid) of the subject. The antigen may also be a membrane antigen.

In further embodiments, the antigen binding activity of the antigen binding domain varies depending on the ionic concentration conditions. In one embodiment, the ion concentration is not particularly limited and relates to hydrogen ion concentration (pH) or metal ion concentration. Herein, the metal ion refers to an ion of a group I element other than hydrogen, such as alkali metals and copper group elements, such as group II elements of alkaline earth metals and zinc group elements, group III elements other than boron, group IV elements other than carbon and silicon, group VIII elements, such as iron group elements and platinum group elements, sub-group a elements belonging to groups V, VI and VII, and metal elements such as antimony, bismuth and polonium. In the present invention, the metal ion includes, for example, a calcium ion as described in WO 2012/073992 and WO 2013/125667. In one embodiment, an "ion concentration condition" may be a condition that focuses on the difference in antigen binding domain biological behavior between a low ion concentration and a high ion concentration. Further, "the antigen binding activity of the antigen binding domain that varies according to the ion concentration condition" means that the antigen binding activity of the antigen binding domain varies between a low ion concentration and a high ion concentration (such antigen binding domain is referred to herein as "ion concentration-dependent antigen binding domain"). The antigen binding activity of the antigen binding domain under high ionic concentration conditions may be higher (stronger) or lower (weaker) than the antigen binding activity under low ionic concentration conditions. In one example, an ionic concentration-dependent antigen-binding domain (e.g., a pH-dependent antigen-binding domain or a calcium ion concentration-dependent antigen-binding domain) can be obtained by known methods, for example, as described in WO 2009/125825, WO 2012/073992, and WO 2013/046722.

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 selected from the following: between 100. mu.M and 10mM, between 200. mu.M and 5mM, between 400. mu.M and 3mM, between 200. mu.M and 2mM, between 400. mu.M and 1mM, or between 500. mu.M and 2.5mM, preferably close to the concentration of plasma (blood) calcium ions in vivo. Meanwhile, the low calcium ion concentration is not particularly limited but may be selected from the following: between 0.1. mu.M and 30. mu.M, between 0.2. mu.M and 20. mu.M, between 0.5. mu.M and 10. mu.M, between 1. mu.M and 5. mu.M or between 2. mu.M and 4. mu.M, preferably close to the calcium ion concentration in early endosomes in vivo.

In one embodiment, the ratio of the antigen-binding activity under the condition of low calcium ion concentration to the antigen-binding activity under the condition of high calcium ion concentration is not limited, but the ratio of the dissociation constant (KD) under the condition of low calcium ion concentration to the KD under the condition of high calcium ion concentration, i.e., KD (low calcium ion concentration condition)/KD (high calcium ion concentration condition), 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, for example, the dissociation rate constant (KD) can be used instead of KD. In this case, the ratio of kd under the condition of low calcium ion concentration to kd under the condition of high calcium ion concentration, i.e., kd (low calcium ion concentration condition)/kd (high calcium ion concentration condition), 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 the antigen binding domain can be generated based on the general technical knowledge of the person skilled in the art.

In the present invention, the antigen binding activity of the antigen binding domain at a low hydrogen ion concentration (neutral pH) may be higher than the antigen binding activity of the antigen binding domain at a high hydrogen ion concentration (acidic pH). The acidic pH may for example be selected from pH4.0 to pH6.5, from pH4.5 to pH6.5, from pH5.0 to pH6.5 or from pH5.5 to pH6.5, preferably close to the in vivo pH in early endosomes. The acidic pH can also be, for example, pH5.8 or pH 6.0. In a particular embodiment, the acidic pH is pH 5.8. Meanwhile, the neutral pH may be selected, for example, from pH6.7 to pH10.0, from pH6.7 to pH9.5, from pH7.0 to pH9.0 or from pH7.0 to pH8.0, preferably close to the in vivo pH in plasma (blood). The neutral pH can also be, for example, pH7.4 or pH 7.0. In a particular embodiment, the neutral pH is pH 7.4.

In one embodiment, the ratio of antigen binding activity under acidic pH conditions and neutral pH conditions is not limited, but the ratio of dissociation constant (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, 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, for example, 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 the antigen binding domain can be generated based on the general technical knowledge of the person skilled in the art.

In one embodiment, for example, at least one amino acid residue is substituted with an amino acid residue having a side chain pKa of 4.0-8.0, and/or at least one amino acid residue having a side chain pKa of 4.0-8.0 is inserted into the antigen binding domain, as described in WO 2009/125825. Amino acids may be substituted and/or inserted at any position as long as the antigen binding activity of the antigen binding domain under acidic pH conditions is weaker than that before the substitution or insertion under neutral pH conditions. When the antigen binding domain has a variable region or CDR, the site may be within the variable region or CDR. The number of substituted or inserted amino acids can be appropriately determined by those skilled in the art; for example, the number may be one or more. Amino acids with side chain pKa's of 4.0-8.0 can be used to alter the antigen binding activity of the antigen binding domain depending on the hydrogen ion concentration conditions. Such amino acids include, for example, natural amino acids such as His (H) and Glu (E), and unnatural amino acids such as histidine analogs (US2009/0035836), 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 pKa's of 6.0-7.0 may also be used, including for example His (H).

In another example, preferred antigen binding domains of variant Fc regions with increased pI are described and may be obtained by the methods described in WO2016/125495 and WO 2017/046994.

In certain embodiments, the variant Fc region having an increased pI comprises at least two amino acid changes 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, according to the EU numbering.

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

In another aspect, the present invention provides a polypeptide comprising a variant Fc region having an increased pI, comprising any one of the following amino acid changes (1) - (10): (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; (10) positions 402 and 413; according to EU numbering.

In one aspect, the invention provides a polypeptide comprising a variant Fc region having enhanced F c γ RIIb binding activity and increased pI, comprising at least three amino acid changes, comprising: (a) at least one amino acid change at least one position selected from the group consisting of: 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, according to EU numbering, and (b) at least two amino acid changes 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, according to the EU numbering.

In one aspect, the invention provides a polypeptide comprising a variant Fc region having enhanced fcyriib binding activity and increased pI, and which comprises at least three amino acid changes, comprising: (a) at least one amino acid change at least one position selected from the group consisting of: 231. 232, 235, 236, 239, 268, 295, 298, 326, 330 and 396, according to EU numbering, and (b) at least two amino acid changes at least two positions selected from the group consisting of: 311. 341, 343, 384, 399, 400, 401, 402 and 413, according to EU numbering.

In another aspect, the present invention provides a polypeptide comprising a variant Fc region having enhanced fcyriib binding activity and increased pI, comprising an amino acid change in any one of the following (1) to (9): (1) positions 235, 236, 268, 295, 311, 326, 330, and 343; (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; (9) positions 236, 268, 330, 396, 400 and 413; according to EU numbering.

In one aspect, the invention provides a polypeptide comprising a variant Fc region having enhanced F c γ RIIb binding activity and increased pI, comprising at least three amino acid changes, comprising: (a) at least one amino acid change 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 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, according to the EU numbering. In other embodiments, the polypeptide comprises at least two amino acid changes at least two positions selected from the group consisting of: 311. 341, 343, 384, 399, 400, 401, 402 and 413, according to EU numbering.

In another aspect, the present invention provides a polypeptide comprising a variant Fc region having enhanced fcyriib binding activity and increased pI, comprising an amino acid change in any one of the following (1) to (16): (1) positions 234, 238, 250, 264, 307, 311, 330, and 343; (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, 307, 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; (16) positions 238, 250, 307, 311, 330, and 413; according to EU numbering.

In addition, amino acid changes made for other purposes may be incorporated into the variant Fc regions described herein. For example, amino acid substitutions that improve FcRn binding activity (Hinton et al, J.Immunol.176(1): 346-. Alternatively, polypeptides having antigen-clearing promoting properties described in WO 2011/122011, WO 2012/132067, WO 2013/046704 or WO 2013/180201, polypeptides having specific binding properties to a target tissue described in WO 2013/180200, polypeptides having the property of repeatedly binding multiple antigen molecules described in WO 2009/125825, WO 2012/073992 or WO 2013/047752 may be combined with the variant Fc regions described herein. Alternatively, the amino acid changes disclosed in EP1752471 and EP1772465 may be combined in CH3 of the variant Fc regions described herein in order to confer binding capacity to other antigens. Alternatively, amino acid changes that decrease the pI of the constant region (WO 2012/016227) can be combined in the variant Fc regions described herein in order to increase plasma retention. Alternatively, amino acid changes that increase the pI of the constant region (WO 2014/145159) can be combined in the variant Fc regions described herein in order to facilitate uptake into cells. Alternatively, amino acid changes that increase the pI of the constant region (WO2016/125495) can be combined in the variant Fc regions described herein in order to facilitate elimination of the target molecule from plasma. In one embodiment, such changes may include, for example, substitution at least one position selected from the group consisting of: 311. 343, 384, 399, 400 and 413, according to EU numbering. In further embodiments, such substitutions may be the substitution of an amino acid with Lys or Arg at each position.

Amino acid changes that enhance human FcRn binding activity at acidic pH can also bind in the variant Fc regions described herein. Specifically, such alterations may include, for example, substitution of Leu for Met at position 428 and Ser for Asn at position 434 (numbering according to EU) (Zalevsky et al, nat. Biotechnol.28:157-159 (2010)); asn at position 434 by Ala (Deng et al, Metab. Dispos.38 (4): 600-605 (2010)); substitution of Met at position 252 with Tyr, Ser at position 254 with Thr, and Thr at position 256 with Glu (Dall' Acqua et al, J.biol.chem.281:23514-23524 (2006)); substitution of Gln for Thr at position 250 and Leu for Met at position 428 (Hinton et al, J.Immunol.176(1):346-356 (2006)); his is substituted at position 434 with Asn (Zheng et al, Clin. Pharmacol. Ther.89(2): 283) -290(2011)), as well as the changes described in 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 2004/035752 or WO 2002/060919. Such changes may include, for example, at least one change selected from the group consisting of: leu for Met at position 428, Ala for Asn at position 434, and Thr for Tyr at position 436. These changes may further comprise a substitution of Arg for Gln at position 438 and/or a substitution of Glu for Ser at position 440 (WO 2016/125495).

Exemplary bispecific anti-CCL 2 antibodies

One embodiment of the invention is a bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second, different antigen-binding site that (specifically) binds to a second, different epitope on human CCL 2.

Wherein the content of the first and second substances,

A) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

Or

B) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

C) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

D) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

E) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

F) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

Or alternatively

G) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

H) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

I) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

In one embodiment, when a pre-formed immune complex consisting of 20mg/kg of each bispecific antibody and 0.1mg/kg of human CCL2 is administered to an FcRn transgenic mouse at a single dose of 10ml/kg, the in vivo clearance (ml/day/kg) of human CCL2 is at least two-fold (in one embodiment, at least 5-fold, in one embodiment, at least 10-fold, in one embodiment, at least 20-fold) higher after administration of a bispecific antibody comprising an fcgamma receptor silencing constant heavy chain domain (or Fc domain thereof) of a human IgG1 isotype comprising mutations L234A, L235A, P329G, compared to the in vivo clearance (ml/day/kg) of human CCL2 after administration of a bispecific antibody comprising a constant heavy chain domain (or Fc domain thereof) of a human wild-type IgG1 isotype.

One embodiment of the 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, different epitope on human CCL2,

wherein the content of the first and second substances,

In one embodiment, when a pre-formed immune complex consisting of 20mg/kg of each bispecific antibody and 0.1mg/kg of human CCL2 is administered to an FcRn transgenic mouse at a single dose of 10ml/kg, the in vivo clearance (ml/day/kg) of human CCL2 after administration of a bispecific antibody comprising an fcgamma receptor silencing constant heavy chain domain of the human IgG1 isotype (or the Fc domain thereof) comprising the mutations L234A, L235A, P329G is at least 15-fold, in particular at least 20-fold higher than the in vivo clearance (ml/day/kg) of human CCL2 after administration of a bispecific antibody comprising a constant heavy chain domain of the human wild-type IgG1 isotype (or the Fc domain thereof).

Wherein the content of the first and second substances,

i) the first antigen binding site comprises

and

and is

ii) the second antigen binding site comprises

and

i) the first antigen binding site comprises

A VH domain comprising: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO 57, wherein X is I or T; (b) CDR-H2 comprising the amino acid sequence GX of SEQ ID NO:58 ¹ IX ² FX ³ TANYAQKFQG, wherein X ¹ Is V, I or H, X ² Is P or H, and X ³ Is H or G; (c) CDR-H3 comprising SEQ ID NO 59 amino acid sequence YDAHYGELDF; (d) FR-H1 comprising the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTF of SEQ ID NO: 63; (e) FR-H2 comprising the amino acid sequence WVRQAPGQGLEWMG of SEQ ID NO 64; (f) FR-H3 comprising the amino acid sequence RVTITADESTSTAYMELSSLRSEDTAVYYCAR of SEQ ID NO: 65; and (g) FR-H4 comprising the amino acid sequence WGQGTLVTVSS of SEQ ID NO: 66;

and

And is

ii) the second antigen binding site comprises

and

wherein the content of the first and second substances,

A) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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

B) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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

C) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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 94;

or

D) 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 is

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 94;

or

E) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 73;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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

F) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 73;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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 94;

or

G) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 73;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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

H) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 73;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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

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 is

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 alternatively

J) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 72;

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

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 is

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

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 is

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

M) i) said 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 is

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 94;

or

N) 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 is

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

O) i) said 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 is

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

P) i) said first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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.

wherein the content of the first and second substances,

i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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;

wherein the content of the first and second substances,

i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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.

wherein the content of the first and second substances,

i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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 94.

wherein the content of the first and second substances,

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 is

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 94.

In one embodiment, the bispecific antibody comprises a constant heavy chain domain of human IgG1 isotype. One embodiment of the 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, different epitope on human CCL2,

A) i) the first antigen binding site comprises

VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO 71, wherein the VH structureThe domain includes: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO 57, wherein X is I; (b) CDR-H2 comprising the amino acid sequence GX of SEQ ID NO:58 ¹ IX ² IFX ³ TANYAQKFQG, wherein X ¹ Is V, X ² Is P, and X ³ Is H; and (c) CDR-H3 comprising amino acid sequence YDAHYGELDF of SEQ ID NO: 59;

ii) the second antigen binding site comprises

or

B) i) the first antigen binding site comprises

(ii) a VH domain sequence,71, wherein the VH domain comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO 57, wherein X is I; (b) CDR-H2 comprising the amino acid sequence GX of SEQ ID NO:58 ¹ IX ² IFX ³ TANYAQKFQG, wherein X ¹ Is V, X ² Is P, and X ³ Is H; and (c) CDR-H3 comprising amino acid sequence YDAHYGELDF of SEQ ID NO: 59;

ii) the second antigen binding site comprises

or

C) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

D) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

and a VL domain sequence having at least 90% amino acid sequence to that of SEQ ID NO 9491%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence KAX of SEQ ID NO:79 ¹ EDIYNRX ² A, wherein, X ¹ Is F or T and X ² Is R or L; (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, wherein X is W or R;

or

E) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

F) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

G) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

H) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

I) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

J) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

K) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

L) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID No. 93, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence KAX of SEQ ID NO:79 ¹ EDIYNRX ² A, wherein, X ¹ Is F or T and X ² Is R or L; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO 80GATSLEH; and (f) CDR-L3 comprising the amino acid sequence QQFXSAPYT of SEQ ID NO. 81, wherein X is W or R;

or

M) i) said first antigen binding site comprises

ii) the second antigen binding site comprises

And a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO 94, wherein the VL isThe domain comprises: (d) CDR-L1 comprising the amino acid sequence KAX of SEQ ID NO:79 ¹ EDIYNRX ² A, wherein, X ¹ Is F or T and X ² Is R or L; (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, wherein X is W or R;

or

N) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

O) i) said first antigen binding site comprises

ii) the second antigen binding site comprises

or

P) i) said first antigen binding site comprises

ii) the second antigen binding site comprises

and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID No. 93, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence KAX of SEQ ID NO:79 ¹ EDIYNRX ² A, wherein, X ¹ Is F or T and X ² Is R or L; (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, wherein X is W or R.

In one embodiment, the bispecific antibodies described herein

iii) cross-react with cynomolgus monkey CCL2 and human CCL 2.

In one embodiment, the bispecific antibody described herein does not cross-react with other CCL homologs, in particular it shows 100-fold less binding to other CCL homologs (selected from the group of CCL8, CCL7, and CCL 13) compared to CCL2 binding.

In one embodiment, the bispecific antibody described herein binds to human CCL2 with 10-fold higher affinity at pH 7.4 than at pH 5.8.

i) S354C and T366W in one of the heavy chain constant domains

ii) Y349C, T366S, L368A, Y407V in the other heavy chain constant domain

In one embodiment, the bispecific antibody comprises a constant heavy chain domain of human IgG1 isotype. In one embodiment, the bispecific antibody described herein comprises a human IgG1 heavy chain constant domain (or Fc domain thereof) comprising one or more of the following mutations (Kabat EU numbering)

iv) Q438R and/or S440E (suitable for inhibiting rheumatoid factor binding).

In one embodiment, the bispecific antibody described herein comprises a human IgG1 heavy chain constant domain (or Fc domain thereof) comprising one or more of the following mutations (Kabat EU numbering)

i) Q311R and/or P343R (adapted to increase pI to enhance antigen uptake); and/or

iv) Q438R and/or S440E (suitable for inhibiting rheumatoid factor binding).

In one embodiment, the bispecific antibody described herein comprises a human IgG1 heavy chain constant domain (or Fc domain thereof), which human IgG1 heavy chain constant domain comprises the following mutations (Kabat EU numbering)

i) Q311R and P343R (adapted to increase 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

iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

In one embodiment, the bispecific antibody described herein comprises a human IgG1 heavy chain constant domain (or Fc domain thereof) comprising the following mutations (Kabat EU numbering)

i) Q311R and P343R (suitable for increasing pI to enhance antigen uptake); and

iii) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

Q311R and P343R (adapted to increase pI to enhance antigen uptake).

iv) Q438R and/or S440E (suitable for inhibiting rheumatoid factor binding).

i) Q311R and P343R (suitable for increasing pI to enhance antigen uptake); and

iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

i) Q311R and P343R (adapted to increase pI to enhance antigen uptake); and

iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

One embodiment of the 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, different epitope on human CCL2, wherein,

A) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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

B) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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

C) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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 94;

or

D) 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 is

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 94;

wherein the bispecific antibody is a full-length antibody of human IgG isotype (preferably human IgG1 isotype)

a) A first light chain and a first heavy chain of a first antibody comprising said first antigen binding site under corresponding i); and

b) a second light chain and a second heavy chain of a second antibody comprising said second antigen-binding site under corresponding ii), and wherein the variable domains VL and VH in the second light chain and the second heavy chain of the second antibody are replaced with each other; and is

Wherein in the constant domain CL of the first light chain under a) the amino acid at position 124 is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) (numbering according to Kabat),

and wherein in the constant domain CH1 of the first heavy chain under a), the amino acid at position 147 is substituted with glutamic acid (E) (numbering according to the Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the Kabat EU index).

i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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;

wherein the bispecific antibody is a (full-length) antibody having

a) A first kappa or lambda light chain and a first heavy chain of the IgG1 isotype comprising the first antigen binding site under i); and

b) a second kappa or lambda light chain and a second IgG1 heavy chain IgG1 isotype comprising the second antigen binding site under ii), and wherein the variable domains VL and VH in the second light chain and second heavy chain of the second antibody are replaced with each other; and

In one embodiment, such bispecific antibodies comprise the following mutations (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

In one embodiment, such bispecific antibodies further comprise the following mutations (Kabat EU numbering):

i) Q311R and P343R (adapted to increase pI to enhance antigen uptake); and

iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

In an alternative embodiment, such bispecific antibodies further comprise the following mutations (Kabat EU numbering):

i) Q311R and P343R (adapted to increase pI to enhance antigen uptake); and

iii) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

Q311R and P343R (adapted to increase pI to enhance antigen uptake).

i) Q311R and P343R (adapted to increase pI to enhance antigen uptake); and

iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

i) Q311R and P343R (adapted to increase pI to enhance antigen uptake); and

iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

A specific embodiment of the 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, wherein the bispecific antibody comprises a heavy chain variable region comprising a heavy chain variable region having a heavy chain variable region (vr) sequence as set forth in SEQ ID NO: 112, an amino acid sequence at least 98% or 99% identical to the sequence of SEQ ID NO: 113, an amino acid sequence which is at least 98% or 99% identical to the sequence of SEQ ID NO: 114, and an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 115, or a polypeptide having an amino acid sequence which is at least 98% or 99% identical.

A particular embodiment of the 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, wherein the bispecific antibody comprises a heavy chain variable region comprising a heavy chain variable region of SEQ ID NO: 112, a polypeptide comprising the amino acid sequence of SEQ ID NO: 113, a polypeptide comprising the amino acid sequence of SEQ ID NO: 114 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 115.

A specific embodiment of the 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, wherein the bispecific antibody comprises a heavy chain variable region comprising a heavy chain variable region having a heavy chain variable region (vr) sequence as set forth in SEQ ID NO: 116, an amino acid sequence at least 98% or 99% identical to the sequence of SEQ ID NO: 117, an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 118, and an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 119, or a polypeptide having an amino acid sequence which is at least 98% or 99% identical to the sequence of 119.

A particular embodiment of the 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, wherein the bispecific antibody comprises a heavy chain variable region comprising a heavy chain variable region of SEQ ID NO: 116, a polypeptide comprising the amino acid sequence of SEQ ID NO: 117, a polypeptide comprising the amino acid sequence of SEQ ID NO: 118 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 119, or a pharmaceutically acceptable salt thereof.

A specific embodiment of the 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, wherein the bispecific antibody comprises a heavy chain variable region comprising a heavy chain variable region having a heavy chain variable region (vr) sequence as set forth in SEQ ID NO: 120, an amino acid sequence at least 98% or 99% identical to the sequence of SEQ ID NO: 121, an amino acid sequence at least 98% or 99% identical to the sequence of SEQ ID NO: 122, and an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 123, or a polypeptide having an amino acid sequence which is at least 98% or 99% identical to the sequence of 123.

A particular embodiment of the 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, wherein the bispecific antibody comprises a heavy chain variable region comprising a heavy chain variable region of SEQ ID NO: 120, a polypeptide comprising the amino acid sequence of SEQ ID NO: 121, a polypeptide comprising the amino acid sequence of SEQ ID NO: 122 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 123.

A specific embodiment of the 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, wherein the bispecific antibody comprises a heavy chain variable region comprising a heavy chain variable region having a heavy chain variable region (vr) sequence as set forth in SEQ ID NO: 120, a polypeptide having an amino acid sequence at least 98% or 99% identical to the sequence of SEQ ID NO: 121, a polypeptide of an amino acid sequence at least 98% or 99% identical to the sequence of SEQ ID NO: 122, and an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 123, or an amino acid sequence which is at least 98% or 99% identical thereto.

A specific embodiment of the 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, wherein the bispecific antibody comprises a heavy chain variable region comprising a heavy chain variable region having a heavy chain variable region (vr) sequence as set forth in SEQ ID NO: 155, an amino acid sequence at least 98% or 99% identical to the sequence of SEQ ID NO: 156, an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 157, and an amino acid sequence at least 98% or 99% identical to the sequence of SEQ ID NO: 158, or a polypeptide having an amino acid sequence which is at least 98% or 99% identical.

A particular embodiment of the 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, wherein the bispecific antibody comprises a heavy chain variable region comprising a heavy chain variable region of SEQ ID NO: 155, a polypeptide comprising the amino acid sequence of SEQ ID NO: 156, a polypeptide comprising the amino acid sequence of SEQ ID NO: 157 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 158.

A specific embodiment of the 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, wherein the bispecific antibody comprises a heavy chain variable region comprising a heavy chain variable region having a heavy chain variable region (vr) sequence as set forth in SEQ ID NO: 159, a polypeptide having an amino acid sequence at least 98% or 99% identical to the sequence of SEQ ID NO: 160, an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 161, and an amino acid sequence at least 98% or 99% identical to the sequence of SEQ ID NO: 162, or a polypeptide having an amino acid sequence which is at least 98% or 99% identical to the sequence of seq id no.

A particular embodiment of the 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, wherein the bispecific antibody comprises a heavy chain variable region comprising a heavy chain variable region of SEQ ID NO: 159, a polypeptide comprising the amino acid sequence of SEQ ID NO: 160, a polypeptide comprising the amino acid sequence of SEQ ID NO: 161 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 162.

A specific embodiment of the 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, wherein the bispecific antibody comprises a heavy chain variable region comprising a heavy chain variable region having a heavy chain variable region (vr) sequence as set forth in SEQ ID NO: 163, an amino acid sequence at least 98% or 99% identical to the sequence of SEQ ID NO: 164, an amino acid sequence at least 98% or 99% identical to the sequence of SEQ ID NO: 165, and an amino acid sequence at least 98% or 99% identical to the sequence of SEQ ID NO: 166, or a polypeptide having an amino acid sequence which is at least 98% or 99% identical in sequence.

A particular embodiment of the 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, wherein the bispecific antibody comprises a heavy chain variable region comprising a heavy chain variable region of SEQ ID NO: 163, a polypeptide comprising the amino acid sequence of SEQ ID NO: 164, a polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 166, or a polypeptide of the amino acid sequence of 166.

A specific embodiment of the 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, wherein the bispecific antibody comprises a heavy chain variable region comprising a heavy chain variable region having a heavy chain variable region (vr) sequence as set forth in SEQ ID NO: 167, an amino acid sequence at least 98% or 99% identical to the sequence of SEQ ID NO: 168, an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 169 having an amino acid sequence at least 98% or 99% identical to the sequence of SEQ ID NO: 170, or a polypeptide having an amino acid sequence which is at least 98% or 99% identical thereto.

A particular embodiment of the 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, wherein the bispecific antibody comprises a heavy chain variable region comprising a heavy chain variable region of SEQ ID NO: 167, a polypeptide comprising the amino acid sequence of SEQ ID NO: 168, a polypeptide comprising the amino acid sequence of SEQ ID NO: 169 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 170, or a pharmaceutically acceptable salt thereof.

A specific embodiment of the 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, wherein the bispecific antibody comprises a heavy chain variable region comprising a heavy chain variable region having a heavy chain variable region (vr) sequence as set forth in SEQ ID NO: 171, an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 172, a polypeptide having an amino acid sequence at least 98% or 99% identical to the sequence of SEQ ID NO: 173, and an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 174, or a polypeptide having an amino acid sequence which is at least 98% or 99% identical to the sequence of seq id no.

A particular embodiment of the 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, wherein the bispecific antibody comprises a heavy chain variable region comprising a heavy chain variable region of SEQ ID NO: 171, a polypeptide comprising the amino acid sequence of SEQ ID NO: 172, a polypeptide comprising the amino acid sequence of SEQ ID NO: 173 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 174, or a polypeptide of the amino acid sequence of seq id no.

i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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 94;

wherein the bispecific antibody is a (full length) antibody of human IgG1 isotype, having

i) S354C and T366W in one of the heavy chain constant domains

ii) Y349C, T366S, L368A, Y407V in the other heavy chain constant domain

i) Q311R and P343R (adapted to increase pI to enhance antigen uptake); and

iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

i) Q311R and P343R (adapted to increase pI to enhance antigen uptake); and

iii) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

Q311R and P343R (adapted to increase pI to enhance antigen uptake).

i) Q311R and P343R (adapted to increase pI to enhance antigen uptake); and

iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

i) Q311R and P343R (adapted to increase pI to enhance antigen uptake); and

iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

A specific embodiment of the 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, wherein the bispecific antibody comprises a heavy chain variable region comprising a heavy chain variable region having a heavy chain variable region (vr) sequence as set forth in SEQ ID NO: 124, an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 125, an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 126, and an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 127, or a polypeptide having an amino acid sequence which is at least 98% or 99% identical to the sequence of seq id no.

A particular embodiment of the 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, wherein the bispecific antibody comprises a heavy chain variable region comprising a heavy chain variable region of SEQ ID NO: 124, a polypeptide comprising the amino acid sequence of SEQ ID NO: 125, a polypeptide comprising the amino acid sequence of SEQ ID NO: 126 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 127.

A specific embodiment of the 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, wherein the bispecific antibody comprises a heavy chain variable region comprising a heavy chain variable region having a heavy chain variable region (vr) sequence as set forth in SEQ ID NO: 128, an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 129, an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 130, and an amino acid sequence at least 98% or 99% identical to the sequence of SEQ ID NO: 131, or a polypeptide having an amino acid sequence which is at least 98% or 99% identical to the sequence of seq id no.

A particular embodiment of the 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, wherein the bispecific antibody comprises a heavy chain variable region comprising a heavy chain variable region of SEQ ID NO: 128, a polypeptide comprising the amino acid sequence of SEQ ID NO: 129, a polypeptide comprising the amino acid sequence of SEQ ID NO: 130 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 131.

A specific embodiment of the 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, wherein the bispecific antibody comprises a heavy chain variable region comprising a heavy chain variable region having a heavy chain variable region (vr) sequence as set forth in SEQ ID NO: 132, an amino acid sequence at least 98% or 99% identical to the sequence of SEQ ID NO: 133, an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 134, and an amino acid sequence at least 98% or 99% identical to the sequence of SEQ ID NO: 135, or a polypeptide having an amino acid sequence which is at least 98% or 99% identical thereto.

A particular embodiment of the 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, wherein the bispecific antibody comprises a heavy chain variable region comprising a heavy chain variable region of SEQ ID NO: 132, a polypeptide comprising the amino acid sequence of SEQ ID NO: 133, a polypeptide comprising the amino acid sequence of SEQ ID NO: 134 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 135, or a pharmaceutically acceptable salt thereof.

Recombinant methods and compositions

Recombinant methods and compositions can be used to produce antibodies, for example, as described in U.S. Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-CCL 2 antibody (bispecific or monospecific) described herein is provided. Such nucleic acids can encode an amino acid sequence comprising one or all VLs and/or an amino acid sequence comprising one or all VH of a monospecific or bispecific antibody (e.g., light and/or heavy chain of an antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (e.g., has been transformed by): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of an antibody and an amino acid sequence comprising a VH of an antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of an antibody, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising a VH of an antibody. In one embodiment, the host cell is eukaryotic, such as Chinese Hamster Ovary (CHO) cells, HEK293 cells, or lymphocytes (e.g., Y0, NS0, Sp20 cells). In one embodiment, a method of making an anti-CCL 2 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as described above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of anti-CCL 2 cells, such nucleic acids can be readily isolated and sequenced using conventional methods (e.g., using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of an antibody).

Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly where glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., US5,648,237, US5,789,199, and US5,840,523. (see also Charlton, K.A., In: Methods In Molecular Biology, vol.248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2003), pp.245-254, which describes the expression of antibody fragments In E.coli (E.coli)), the antibodies can be separated from the soluble fraction of the bacterial cell paste and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms (e.g., filamentous fungi or yeast) are also suitable hosts for the cloning or expression of polypeptide-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of polypeptides having a partially or fully human glycosylation pattern. See Gerngross, T.U., nat. Biotech.22(2004) 1409-; and Li, H. et al, nat. Biotech.24(2006) 210-.

Suitable host cells for expression of glycosylated polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified which can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells.

Plant cell cultures may also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing antibody-producing PLANTIBODIIES in transgenic plants ^TM A technique).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be used. Other examples of mammalian host cell strains that may be used are: monkey kidney CV1 cell line transformed with SV40 (COS-7); human embryonic kidney cell lines (293 or 293T cells as described in Graham, F.L. et al, J.Gen Virol.36(1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, J.P., biol. reprod.23(1980)243- > 252); monkey kidney cells (CV 1); VERO cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells (as described in Mather, J.P. et al, Annals N.Y.Acad.Sci.383(1982) 44-68); MRC 5 cells; and FS4 cells. Other mammalian host cell lines that may be used include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al, Proc. Natl. Acad. Sci. USA 77(1980) 4216-4220); and myeloma cell lines, such as Y0, NS0, and Sp 2/0. For reviews of certain mammalian host cell lines suitable for antibody production, see, for example: yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol.248, Lo, B.K.C. Main eds, Humana Press, Totowa, NJ (2004), p.255-268).

In another aspect, the invention is based, in part, on the following findings: the modified monospecific antibodies described herein exhibit improved pH-dependent binding properties and are therefore particularly useful for the generation of bispecific antibodies of the invention.

Monospecific anti-CCL 2 antibodies with pH dependent binding properties

wherein the antibody comprises

and

or

And

wherein the antibody comprises

A) A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75;

or

B) 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;

or

C) A VH domain comprising the amino acid sequence of SEQ ID NO 73;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75;

or

D) 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;

or

E) 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

F) 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

G) 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

H) 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 94;

methods and compositions for diagnosis and detection

In certain embodiments, any of the monospecific or bispecific anti-CCL 2 antibodies provided herein are useful for detecting the presence of CCL2 in a biological sample. The term "detecting", as used herein, encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues, such as immune cells or T cell infiltrates and/or tumor cells.

In one embodiment, a monospecific or bispecific anti-CCL 2 antibody is provided for use in a diagnostic or detection method. In another aspect, a method of detecting the presence or absence of CCL2 in a biological sample is provided. In certain embodiments, the method comprises contacting a biological sample with a monospecific or bispecific anti-CCL 2 antibody described herein under conditions that allow binding of the monospecific or bispecific anti-CCL 2 antibody to CCL2, and detecting whether a complex is formed between the monospecific or bispecific anti-CCL 2 antibody and CCL 2. Such methods may be in vitro or in vivo. In one embodiment, a monospecific or bispecific anti-CCL 2 antibody is used to select subjects suitable for treatment with a monospecific or bispecific anti-CCL 2 antibody, e.g., wherein CCL2 is a biomarker for selecting patients.

In certain embodiments, a labeled monospecific or bispecific anti-CCL 2 antibody is provided. Labels include, but are not limited to, labels or moieties that are directly detectable (e.g., fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), as well as moieties that are indirectly detectable (e.g., by enzymatic reactions or molecular interactions), such as enzymes or ligands. Display deviceExemplary markers include, but are not limited to: radioisotope ³² P、 ¹⁴ C、 ¹²⁵ I、 ³ H and ¹³¹ i; fluorophores such as rare earth chelates or fluorescein and its derivatives; rhodamine and its derivatives, dansyl, umbelliferone; luciferases, such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456); fluorescein; 2, 3-dihydronaphthyridine dione; horseradish peroxidase (HRP); alkaline phosphatase; beta-galactosidase; a glucoamylase; lysozyme; carbohydrate oxidases such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase; heterocyclic oxidases such as urate oxidase and xanthine oxidase; coupled with an enzyme that oxidizes a dye precursor with hydrogen peroxide (e.g., HRP, lactoperoxidase, or microperoxidase); biotin/avidin; marking the spinning; phage labels or stabilizes free radicals.

E. Pharmaceutical preparation

Pharmaceutical formulations of monospecific or bispecific anti-CCL 2 antibodies described herein are prepared in lyophilized formulations or in aqueous solution by mixing an antibody of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16 th edition, Osol, a. (ed.) (1980)). Pharmaceutically acceptable carriers, which are generally non-toxic to recipients at the dosages and concentrations employed, include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (for example octadecyl dimethyl benzyl ammonium chloride; hexa-hydrocarbonic quaternary ammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, for example methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or pastes Refining; chelating agents (e.g., EDTA saccharides); sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc protein complexes); and/or a non-ionic surfactant, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers in the present application further include interstitial drug dispersing agents, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, such as rhuPH20 (h: (h))Baxter International, Inc.). Certain exemplary shasegps and uses, including rhuPH20, are described in U.S. patent publication nos. 2005/0260186 and 2006/0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Water-soluble antibody formulations include those described in U.S. patent No. 6,171,586 and WO 2006/044908, the latter formulations including histidine-acetate buffer.

The formulations described herein may also comprise more than one active ingredient suitable for the particular indication being treated, preferably those having complementary active ingredients that do not adversely affect each other. For example, further provisioning may be required. These active ingredients are suitably present in combination in an amount effective for the intended purpose.

The active ingredient may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose microcapsules or gelatin-and poly (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. This technique is disclosed in Remington's Pharmaceutical Sciences, 16 th edition, Osol, a. (ed.) (1980).

Sustained release formulations can be prepared. Suitable examples of delivery formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.

Formulations for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

F. Therapeutic methods and compositions

Any of the monospecific or bispecific anti-CCL 2 antibodies provided herein can be used in a method of treatment.

In one aspect, a monospecific or bispecific anti-CCL 2 antibody for use as a medicament is provided. In other aspects, a monospecific or bispecific anti-CCL 2 antibody for use in treating cancer is provided. In certain embodiments, a monospecific or bispecific anti-CCL 2 antibody is provided for use in a method of treatment. In certain embodiments, the present invention provides a method of treating an individual having cancer with a monospecific or bispecific anti-CCL 2 antibody, comprising administering to the individual a therapeutically effective amount of a monospecific or bispecific anti-CCL 2 antibody.

In further embodiments, the invention provides monospecific or bispecific anti-CCL 2 antibodies that inhibit immunosuppression in tumors, and thus sensitize tumors to immunostimulants such as anti-PD 1, anti-PDL-1 antagonists, and the like.

Thus, one aspect thereof is a combination of a monospecific or bispecific anti-CCL 2 antibody described herein with a cancer immunotherapy, e.g., anti-PD 1, anti-PDL-1 antagonist, etc.

As used herein, the term "cancer" may be, for example, lung cancer, non-small cell lung cancer (NSCL), bronchoalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, cancer of the prostate, cancer of the bladder, cancer of the kidney or ureter, cancer of the renal cells, carcinoma of the renal pelvis, mesothelioma, hepatocellular carcinoma, cancer of the bile duct, tumors of the Central Nervous System (CNS), tumors of the spinal axis, glioma of the brain stem, glioblastoma multiforme, astrocytoma, schwannoma, neuroblastoma, head tumor, cancer of the head of the, Ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia, refractory forms including any of the foregoing, or combinations of one or more of the foregoing.

The "individual" according to any of the above embodiments is preferably a human. In another aspect, the invention provides the use of a monospecific or bispecific anti-CCL 2 antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treating cancer. In another embodiment, the medicament is for use in a method of treating cancer, the method comprising administering to an individual having cancer a therapeutically effective amount of the medicament. In another embodiment, the medicament is for inducing cell-mediated lysis of cancer cells. In another embodiment, the medicament is for use in a method of inducing cell-mediated lysis of cancer cells in an individual having cancer, the method comprising administering to the individual an effective amount of the medicament to induce apoptosis in the cancer cells and/or inhibit proliferation of the cancer cells. An "individual" according to any of the embodiments above may be a human.

In another aspect, the invention provides a method of treating cancer. In one embodiment, the method comprises administering to an individual having cancer an effective amount of a monospecific or bispecific anti-CCL 2 antibody. An "individual" according to any of the embodiments above may be a human.

In another aspect, the invention provides a method of inducing cell-mediated lysis of cancer cells in an individual having cancer. In one embodiment, the method comprises administering to an individual an effective amount of a monospecific or bispecific anti-CCL 2 antibody to induce cell-mediated lysis of cancer cells in an individual having cancer. In one embodiment, the "individual" is a human.

In another aspect of the invention, a monospecific or bispecific anti-CCL 2 antibody is provided for use in the treatment of an inflammatory disease or an autoimmune disease. In certain embodiments, the present invention provides a method of treating an individual having an inflammatory or autoimmune disease with a monospecific or bispecific anti-CCL 2 antibody, comprising administering to the individual a therapeutic amount of a monospecific or bispecific anti-CCL 2 antibody.

In another aspect, the invention provides a pharmaceutical formulation comprising any of the monospecific or bispecific anti-CCL 2 antibodies provided herein, e.g., for use in any of the methods of treatment above. In one embodiment, a pharmaceutical formulation comprises any of the monospecific or bispecific anti-CCL 2 antibodies provided herein and a pharmaceutically acceptable carrier.

In some embodiments, the inflammatory or autoimmune disease is an autoimmune disease, an inflammatory disease, a fibrotic disease, a myelogenous (neutrophilic or eosinophilic) disease, a monocytic or lymphocytic disease, or a condition associated with an increase in the number or distribution of normal or abnormal tissue resident cells (e.g., mast cells, macrophages or lymphocytes) or stromal cells (e.g., fibroblasts, myofibroblasts, smooth muscle cells, epithelial cells, or endothelial cells). In some embodiments, the disorder is a pulmonary disorder. In some embodiments, the pulmonary disorder is one associated with granulocytic (eosinophilic and/or neutrophilic) pulmonary inflammation, infection-induced pulmonary symptoms (including those associated with viral (e.g., influenza, parainfluenza, rhinovirus, human interstitial pneumovirus, and respiratory syncytial virus), bacterial or fungal (e.g., aspergillus) triggers Lymphangiosarcoidosis, acute lung injury, acute respiratory distress syndrome, chronic lung disease, bronchopulmonary dysplasia, pneumonia (e.g., community-acquired pneumonia, hospital-acquired pneumonia, ventilator-associated pneumonia, viral pneumonia, bacterial pneumonia, and severe pneumonia), respiratory exacerbations, and Acute Respiratory Distress Syndrome (ARDS). In some embodiments, the inflammatory lung disease is COPD.

In some embodiments, the inflammatory lung disease is asthma. In some embodiments, asthma is persistent chronic severe asthma with an acute attack of potentially life-threatening exacerbating symptoms (exacerbations or attacks). In some embodiments, the asthma is atopic (also referred to as allergic) asthma, non-allergic asthma (e.g., often caused by infection with respiratory viruses (e.g., influenza, parainfluenza, rhinovirus, human metapneumovirus, and respiratory syncytial virus), or inhalation irritants (air pollutants, smoke, diesel particulates, volatile chemicals, and gases in indoor or outdoor and even cold dry air).

In some embodiments, asthma is intermittent or exercise-induced asthma, asthma resulting from acute or chronic primary or secondary exposure to second-hand "smoke" (typically cigarettes, cigars, pipes), inhalation or "inhalation" (tobacco, cannabis or other substances), or asthma resulting from recent intake of aspirin or related NSAIDS. In some embodiments, the asthma is mild asthma or corticosteroid incipient asthma, newly diagnosed and untreated asthma, or is symptomatic (cough, wheezing, shortness of breath/dyspnea, or chest pain) with local or systemic steroids that previously did not require prolonged use of inhalation. In some embodiments, the asthma is chronic, corticosteroid-resistant asthma, corticosteroid-refractory asthma, asthma uncontrolled by corticosteroids or other chronic asthma-controlling drugs. In some embodiments, the autoimmune disease, inflammatory disease, fibrotic disease, neutrophil disease, or eosinophilic disease is pulmonary fibrosis. In some embodiments, the pulmonary fibrosis is Idiopathic Pulmonary Fibrosis (IPF). In some embodiments, the autoimmune disease, inflammatory disorder, fibrotic disorder, myelogenous (neutrophilic or eosinophilic) disorder, monocytic or lymphocytic disorder is esophagitis, allergic rhinitis, non-allergic rhinitis, sinusitis with nasal polyps, bronchitis, chronic pneumonia, allergic bronchopulmonary aspergillosis, airway inflammation, allergic rhinitis, bronchiectasis, and/or chronic bronchitis.

In some embodiments, the autoimmune disease, inflammatory disease, fibrotic disease, myelogenous (neutrophilic or eosinophilic) disease, monocytic disease or lymphocytic disease is arthritis. In some embodiments, the arthritis is rheumatoid arthritis. In some embodiments, the arthritis is osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, early stage arthritis, polyarticular rheumatoid arthritis, systemic onset rheumatoid arthritis, enteropathic arthritis, reactive arthritis, psoriatic arthritis, and/or arthritis caused by injury.

In some embodiments the autoimmune disease, inflammatory disease, fibrotic disease, myelogenous (neutrophilic or eosinophilic) disease, monocytic disease or lymphocytic disease is a gastrointestinal inflammatory disease. In some embodiments, the gastrointestinal inflammatory disease is IBD (inflammatory bowel disease), Ulcerative Colitis (UC), Crohn's Disease (CD), colitis (e.g., colitis caused by an environmental insult (e.g., caused by or associated with a treatment regimen, such as chemotherapy, radiation therapy, etc.), infectious colitis, ischemic colitis, collagenous or lymphocytic colitis, necrotizing enterocolitis, colitis caused by gastrointestinal inflammation due to infectious diseases, such as chronic granulomatous disease or celiac disease, food allergies, gastritis, gastroenteritis, infectious gastritis or enterocolitis (e.g., chronic active gastritis infected with helicobacter pylori), and other gastrointestinal inflammation caused by infectious diseases, or indeterminate colitis.

In some embodiments, the autoimmune disease, inflammatory disease, fibrotic disease, myelogenous (neutrophilic or eosinophilic) disease, monocytic or lymphocytic disease, or cell resident with normal or abnormal tissue (e.g., mast cells, macrophages or lymphocytes) or stromal cells (e.g., fibroblasts, myofibroblasts, smooth muscle cells, epithelial cells or endothelial cells) is Lupus or Systemic Lupus Erythematosus (SLE), or an organ-specific manifestation of one or more Lupus (e.g., Lupus Nephritis (LN) affecting the kidney), or an organ-specific manifestation of one or more Lupus (lymph nodes, spleen, thymus and associated lymphatic vessels) and/or joints and/or other organs, but not necessarily extrarenal Lupus (ERL)). In some embodiments, the autoimmune, inflammatory, or fibrotic disease is related to sepsis and/or trauma, HIV infection, or idiopathic (etiology unknown) such as ANCA-associated vasculitis (AAV), granulomatous polyangiitis (formerly known as Wegener's granulomatosis), Behcet's disease, cardiovascular disease, eosinophilic bronchitis, retter's Syndrome, SEA Syndrome (seronegative, enteropathy, arthritic Syndrome), ankylosing spondylitis, dermatomyositis, scleroderma, e.g., systemic scleroderma (also known as systemic sclerosis), vasculitis (e.g., Giant Cell Arteritis (GCA), also known as temporal Arteritis, craniitis, or hordon), myositis, polymyositis, dermatomyositis, nodular multiple Arteritis, or combinations thereof), Rheumatic myalgia, sarcoidosis, primary biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis, reverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, pemphigus (e.g. pemphigus vulgaris), atherosclerosis, lupus, still's disease, myasthenia gravis, celiac disease, relapsing-remitting (RRMS) or Primary Progressive (PPMS) or Secondary Progressive (SPMS) subtypes of multiple sclerosis (Ms), Guilin-Barre (Guillain-Barre) disease, type I diabetes (T1DM) or insulin-dependent (IDDM) or juvenile DM, thyroiditis (e.g. Graves ' disease), celiac disease, Chager-Strause syndrome, myalgia syndrome, cytosis syndrome, angioedema reactions including paroxysmal edema, Helminthic infection, dermatitis disclosutia, eosinophilic esophagitis, eosinophilic enteritis, eosinophilic colitis, obstructive sleep apnea, endocardial fibrosis, Addison's (Addison) disease, Raynaud's (Raynaud) disease or phenomenon, autoimmune hepatitis, Graft Versus Host Disease (GVHD) or organ transplant rejection.

The immune complexes (and any other therapeutic agents) of the invention may be administered by any suitable means, including parenteral, intrapulmonary and intranasal administration, and if topical treatment is required, intralesional administration may be employed. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example by injection, for example intravenous or subcutaneous injection, depending in part on whether the administration is brief or chronic. Various dosing regimens are contemplated herein, including, but not limited to, single or multiple administrations at different time points, bolus administration, and pulse infusion.

The antibodies of the invention will be formulated, administered and administered in a manner consistent with good medical practice. Factors considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to medical practitioners. The antibody need not be, but may optionally be formulated with one or more agents currently used for the prevention or treatment of the disease. The effective amount of these other drugs will depend on the amount of antibody present in the formulation, the type of disease or treatment, and other factors discussed above. These agents are generally used at the same dosages and routes of administration as described herein, or from about 1% to 99% of the dosages described herein, or at any dosage and by any route empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of an antibody of the invention (either alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, the administration of the antibody for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient in one or a series of treatments. Depending on the type and severity of the disease, about 1. mu.g/kg to 15mg/kg (e.g., 0.5mg/kg-10mg/kg) of antibody may be an initial candidate dose for administration to a patient, whether, for example, by one or more separate administrations, or by continuous infusion. Depending on the factors described above, a typical daily dose may range from about 1 μ g/kg to 100mg/kg or more. For repeated administration over several days or longer, depending on the condition, treatment will generally continue until the desired suppression of disease symptoms occurs. An exemplary dose of antibody will range from about 0.05mg/kg to about 10 mg/kg. Thus, a patient may be administered a dose of one or more of about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg, or 10mg/kg (or any combination thereof). Such doses may be administered intermittently, such as weekly or every three weeks (e.g., to subject the patient to about 2 to about 20 or.e., about 6 doses of antibody). An initial higher loading dose may be administered followed by one or more lower doses. An exemplary dosing regimen comprises administering an initial loading dose of about 4mg/kg followed by a weekly maintenance dose of about 2mg/kg of antibody. However, other dosage regimens may be used. The progress of such treatment is readily detected by conventional techniques and assays.

II. product

Another aspect of the invention provides an article of manufacture comprising a compound for the treatment, prevention and/or diagnosis of the above-mentioned diseases. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, Intravenous (IV) solution bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container may contain a composition, either by itself or in combination with another composition effective for treating, preventing and/or diagnosing a disease, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is for use in treating the selected disease. In addition, the article of manufacture can include (a) a first container having a composition therein, wherein the composition comprises an antibody of the invention; and (b) a second container having a composition contained therein, wherein the composition contains an additional cytotoxic or other therapeutic agent. The article of manufacture of this embodiment of the invention may further comprise package inserts indicating that the composition may be used to treat a particular disease. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. From a commercial and user perspective, it may further comprise other materials, including other buffers, diluents, filters, needles and syringes.

Listed in the following specific examples of the invention:

1. a bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second, different antigen-binding site that (specifically) binds to a second, different epitope on human CCL2, wherein the bispecific antibody comprises an Fc domain of a human IgG isotype, preferably of the IgG1 isotype.

2. According to the bispecific antibody of example 1,

wherein the content of the first and second substances,

A) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

B) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

Or alternatively

C) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

D) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

E) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

F) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

G) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

Or alternatively

H) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

I) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

3. According to the bispecific antibody of example 2,

wherein the content of the first and second substances,

A) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

and a VL domain comprising the amino acid sequence of SEQ ID NO 48, wherein the VL domain comprises: (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;

or

B) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 23, wherein the VH domain comprises: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO 17; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO. 18; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO. 19;

and a VL domain comprising the amino acid sequence of SEQ ID NO 24, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO. 20; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO 21; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO. 22;

or

C) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 15, wherein the VH domain comprises: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO 9; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO. 10; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO. 11;

and a VL domain comprising the amino acid sequence of SEQ ID NO 16, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO. 12; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO 13; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO. 14;

or

D) i) the first antigen binding site comprises

and a VL domain comprising the amino acid sequence of SEQ ID NO 24, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO. 20; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO 21; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO. 22; and is

ii) the second antigen binding site comprises

or

E) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 31, wherein the VH domain comprises: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO. 25; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO. 26; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27;

and a VL domain comprising the amino acid sequence of SEQ ID NO 32, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO 28; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 29; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30; and is

ii) the second antigen binding site comprises

or

F) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO:55, wherein the VH domain comprises: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO. 49; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO 50; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 51;

and a VL domain comprising the amino acid sequence of SEQ ID NO 56, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO 52; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO 53; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; and is

ii) the second antigen binding site comprises

or

G) i) the first antigen binding site comprises

and a VL domain comprising the amino acid sequence of SEQ ID NO 16, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO. 12; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO 13; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO. 14; and is

ii) the second antigen binding site comprises

or

H) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

and a VL domain comprising the amino acid sequence of SEQ ID NO 32, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO 28; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 29; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30;

or

I) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 7, wherein the VH domain comprises: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO 1; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO. 2; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO. 3;

and a VL domain comprising the amino acid sequence of SEQ ID NO 8, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO. 4; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO 5; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO 6; and is

ii) the second antigen binding site comprises

and a VL domain comprising the amino acid sequence of SEQ ID NO 32, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO 28; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 29; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

4. The bispecific antibody of any one of claims 1 to 3, wherein the bispecific antibody comprising an Fc domain of a human IgG isotype is a bispecific antibody comprising a constant heavy chain domain of a human IgG1 isotype.

5. The bispecific antibody according to example 4, wherein the in vivo clearance (ml/day/kg) of human CCL2 is at least two times (in one embodiment at least 5 times higher, in one embodiment at least 10 times higher, in one embodiment at least 20 times higher) after administration of a bispecific antibody comprising a constant heavy chain domain of human wild-type IgG1 isotype, compared to the in vivo clearance (ml/day/kg) of human CCL2 after administration of a bispecific antibody comprising an fcgamma receptor silent constant heavy chain domain of human IgG1 isotype comprising mutations L234A, L235A, P329G (Kabat EU numbering) when a pre-formed immune complex consisting of 20mg/kg of each bispecific antibody and 0.1mg/kg of human CCL2 is administered at a single dose of 10 ml/kg.

6. 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, different epitope on human CCL2,

wherein the bispecific antibody comprises an Fc domain of a human IgG isotype, wherein,

7. The bispecific antibody according to example 6, wherein the in vivo clearance (ml/day/kg) of human CCL2 after administration of the bispecific antibody comprising a constant heavy chain domain of the human wild-type IgG1 isotype is at least 15-fold (in particular at least 20-fold) higher than the in vivo clearance (ml/day/kg) of human CCL2 after administration of a bispecific antibody comprising an Fc γ receptor silent constant heavy chain domain of the human IgG1 isotype comprising the mutations L234A, L235A, P329G (Kabat EU numbering), when a preformed immune complex consisting of 20mg/kg of each bispecific antibody and 0.1mg/kg of human CCL2 is administered to an FcRn transgenic mouse at a single dose of 10 ml/kg.

8. 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,

Wherein the content of the first and second substances,

i) the first antigen binding site comprises

and

and is

ii) the second antigen binding site comprises

and

9. 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,

wherein the content of the first and second substances,

i) the first antigen binding site comprises

and

And is provided with

ii) the second antigen binding site comprises

and

10. 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 comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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

B) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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

C) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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 94; or

D) 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 is

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 94; or

E) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 73;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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

F) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 73;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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 94; or

G) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 73;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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

H) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 73;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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

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 is

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 alternatively

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 is

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

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 is

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

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 is

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 alternatively

M) i) said first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 74;

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 94;

or

N) 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 is

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

O) i) said 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 is

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

P) i) said first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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.

11. 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,

wherein the content of the first and second substances,

A) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

and VL structureA domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO 93, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence KAX of SEQ ID NO:79 ¹ EDIYNRX ² A, wherein, X ¹ Is F and X ² Is R; (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, wherein X is W;

or

B) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

C) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

D) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

E) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

F) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

G) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

H) i) the first antigen binding site comprises

VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence to the amino acid sequence of SEQ ID NO. 73 Wherein the VH domain comprises: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO 57, wherein X is I or T; (b) CDR-H2 comprising the amino acid sequence GX of SEQ ID NO:58 ¹ IX ² IFX ³ TANYAQKFQG, wherein X ¹ Is V, I or H, X ² Is P or H, and X ³ Is H or G; and (c) CDR-H3 comprising amino acid sequence YDAHYGELDF of SEQ ID NO: 59;

ii) the second antigen binding site comprises

or

I) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID No. 93, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence KAX of SEQ ID NO:79 ¹ EDIYNRX ² A, wherein, X ¹ Is F or T and X ² Is R or L; (e) CDR-L2 comprising SEQ ID NO 80The amino acid sequence GATSLEH; and (f) CDR-L3 comprising the amino acid sequence QQFXSAPYT of SEQ ID NO. 81, wherein X is W or R;

or

J) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

And a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:93,wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence KAX of SEQ ID NO:79 ¹ EDIYNRX ² A, wherein, X ¹ Is F or T and X ² Is R or L; (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, wherein X is W or R;

or

K) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO 91, wherein the VH domain comprises: (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, wherein X is D or E; and (c) CDR-H3 comprising the amino acid sequence GVGFFXH of SEQ ID NO. 78, wherein X is D or E;

or

L) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

M) i) said first antigen binding site comprises

ii) the second antigen binding site comprises

or

N) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

O) i) said first antigen binding site comprises

ii) the second antigen binding site comprises

or

P) i) said first antigen binding site comprises

ii) the second antigen binding site comprises

12. The bispecific antibody according to any one of embodiments 8 to 11, wherein the bispecific antibody is an Fc domain of a human IgG isotype, preferably of the human IgG1 isotype.

13. The bispecific antibody according to any one of embodiments 8 to 11, wherein the bispecific antibody comprises a constant domain of a human IgG isotype, preferably of the human IgG1 isotype.

14. The bispecific antibody according to any one of the preceding embodiments, wherein the bispecific antibody

iii) cross-reacts with cynomolgus macaque and human CCL 2.

15. The bispecific antibody of any one of embodiments, wherein the bispecific antibody has no cross-reactivity with other CCL homologs (showing a 100-fold reduction in binding to other CCL homologs (selected from the group of CCL8, CCL7, and CCL 13) compared to binding CCL 2.

16. The bispecific antibody according to any one of the preceding embodiments, wherein the bispecific antibody binds to a first and a second epitope on human CCL2 in an ion-dependent manner.

17. The bispecific antibody according to any one of the preceding embodiments, wherein the bispecific antibody binds human CCL2 in a pH-dependent manner, and wherein both the first antigen-binding site and the second antigen-binding site bind CCL2 with higher affinity at neutral pH than at acidic pH.

18. The bispecific antibody according to any one of the preceding embodiments, wherein the bispecific antibody binds human CCL2 with 10-fold higher affinity at pH 7.4 than at pH 5.8.

19. The bispecific antibody according to any one of the preceding claims, wherein the bispecific antibody comprises a human IgG1 heavy chain constant domain comprising the following mutations (one or more of Kabat EU numbering (s))

ii) L234Y, L235W, G236N, P238D, T250V, V264I, T307PH268D, Q295L, K326T and/or a330K (suitable for increasing affinity to human FcgRIIb and to other human FcgR); and/or

iii) M428L, N434A and/or Y436T (suitable for increasing affinity for FcRn to prolong plasma half-life); and/or

iv) Q438R and/or S440E (suitable for inhibiting rheumatoid factor binding).

20. The bispecific antibody according to any one of the preceding embodiments, wherein the bispecific antibody comprises a human IgG1 comprising one or more of the following mutations (Kabat EU numbering)

Heavy chain constant domains

iv) Q438R and/or S440E (suitable for inhibiting rheumatoid factor binding).

21. The bispecific antibody according to any one of the preceding embodiments, wherein the bispecific antibody comprises a human IgG1 heavy chain constant domain comprising the following mutations (Kabat EU numbering)

i) Q311R and P343R (adapted to increase pI to enhance antigen uptake); and

iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

22. The bispecific antibody according to any one of the preceding claims, wherein the bispecific antibody comprises a human IgG1 heavy chain constant domain comprising the following mutations (one or more of Kabat EU numbering (s))

ii) L234Y, P238D, T250V, V264I, T307V and/or a330K (suitable for increasing affinity to human FcgRIIb and to other human FcgR); and/or

iv) Q438R and/or S440E (suitable for inhibiting rheumatoid factor binding).

23. The bispecific antibody according to any one of the preceding claims, wherein the bispecific antibody comprises a human IgG1 heavy chain constant domain comprising the following mutations (one or more of Kabat EU numbering (s))

i) Q311R and P343R (adapted to increase pI to enhance antigen uptake); and

ii) L234Y, P238D, T250V, V264I, T307V and a330K (suitable for increasing affinity to human FcgRIIb and to other human FcgR); and

iii) M428L, N434A and Y436T (suitable for increasing affinity for FcRn to prolong plasma half-life); and

iv) Q438R and S440E (suitable for inhibiting rheumatoid factor) binding.

24. The bispecific antibody according to any one of the preceding claims, wherein the bispecific antibody comprises a human IgG1 heavy chain constant domain comprising the following mutations (one or more of Kabat EU numbering (s))

i) Q311R and P343R (adapted to increase pI to enhance antigen uptake); and

ii) L234Y, P238D, T250V, V264I, T307V and a330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and

iv) Q438R and S440E (suitable for inhibiting rheumatoid factor) binding.

25. The bispecific of any one of the preceding embodiments, wherein the bispecific antibody comprises two human IgG1 heavy chain constant domains comprising the following mutations (EU numbering)

i) S354C and T366W in one of the heavy chain constant domains

ii) Y349C, T366S, L368A, Y407V26 in the other heavy chain constant domain an (isolated) antibody which binds (specifically) to human CCL2,

wherein the antibody comprises

and

or

and

27. An (isolated) antibody that binds (specifically) to human CCL2,

wherein the antibody comprises

A) A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75;

or

B) 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;

or

C) A VH domain comprising the amino acid sequence of SEQ ID NO 73;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75;

or

D) 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;

or

E) 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

F) 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 alternatively

G) 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

H) 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 94;

28. an isolated nucleic acid encoding an antibody according to any one of the preceding embodiments.

29. A host cell comprising the nucleic acid of example 28.

30. A method of producing an antibody comprising culturing the host cell of example 29, thereby producing the antibody.

31. The method of embodiment 30, further comprising recovering the antibody from the host cell.

32. A pharmaceutical formulation comprising a bispecific antibody according to any one of embodiments 1 to 25 and a pharmaceutically acceptable carrier.

33. A bispecific antibody according to any one of embodiments 1 to 25 for use as a medicament.

34. The bispecific antibody according to any one of embodiments 1 to 25 for use in the treatment of cancer.

35. A bispecific antibody according to any one of embodiments 1 to 25 for use in the treatment of an inflammatory or autoimmune disease.

36. Use of a bispecific antibody according to any one of embodiments 1 to 25 in the manufacture of a medicament.

37. The use of embodiment 36, wherein the medicament is for the treatment of cancer.

38. The use of example 36, wherein the medicament is for treating an inflammatory or autoimmune disease.

39. A method of treating an individual having cancer comprising administering to the individual an effective amount of a bispecific antibody according to any one of embodiments 1 to 25.

40. A method of treating an individual having an inflammatory or autoimmune disease comprising administering to the individual an effective amount of a bispecific antibody according to any one of examples 1 to 25.

The following examples and drawings are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It will be appreciated that modifications may be made to the proposed method without departing from the spirit of the invention.

Description of the amino acid sequence

anti-CCL 2 antigen-binding portions (variable and hypervariable regions (CDRs)) that bind to different epitopes:

CDR-modified anti-CCL 2 antigen-binding portions (variable and hypervariable regions (CDRs)):

modified CNTO888

Modified humanized 11K2

Exemplary constant light chain regions:

exemplary human kappa light chain constant region of SEQ ID NO 95

96 exemplary human Lambda light chain constant region of SEQ ID NO

Exemplary constant heavy chain regions:

97 exemplary human heavy chain constant region derived from IgG1

98 derived from IgG1 an exemplary human heavy chain constant region with mutations L234A, L235A, and P329G (Fc γ receptor silencing)

99 exemplary human heavy chain constant region derived from IgG1 (SG1-IgG1 allotype) SEQ ID NO: 100 exemplary human heavy chain constant region derived from IgG1 with mutations (SG105-IgG1 allotype-Fc γ receptor silencing)

101SG 1095-an exemplary human heavy chain constant region derived from IgG1, including mutations (Kabat EU numbering):

L235W/G236N/H268D/Q295L/a330K/K326T (suitable for increasing the affinity to human FcgRIIb and to other human FcgR);

-Q311R/P343R (adapted to increase the isoelectric point (pI) to enhance antigen uptake;

-N434A (suitable for increasing the affinity for FcRn to prolong the plasma half-life of the antibody; and

-Q438R/S440E (suitable for inhibiting rheumatoid factor binding)

102SG 1099-an exemplary human heavy chain constant region derived from IgG1, including mutations (Kabat EU numbering):

Q311R/P343R (adapted to increase pI to enhance antigen uptake)

103SG 1100-an exemplary human heavy chain constant region derived from IgG1, including mutations (Kabat EU numbering):

-Q311R/P343R (adapted to increase pI to enhance antigen uptake);

-N434A (suitable for increasing affinity to FcRn to prolong the plasma half-life of the antibody); and

-Q438R/S440E (adapted to inhibit rheumatoid factor binding) CNTO888//11K2-WT IgG1 (exemplary bispecific CNTO888//11K2-WT IgG1 Crossmab)

SEQ ID NO 104 heavy chain 1-CNTO888//11K2-WT IgG1

105 heavy chain 2-CNTO888//11K2-WT IgG1 of SEQ ID NO

106 light chain 1-CNTO888//11K2-WT IgG1 of SEQ ID NO

107 light chain 2-CNTO888//11K2-WT IgG1 of SEQ ID NO:107

CKLO 2-IgG 1 (exemplary bispecific CKLO2 IgG1 Crossmab)

108 heavy chain 1-CKLO2 IgG1 SEQ ID NO

109 heavy chain 2-CKLO2 IgG1 SEQ ID NO

110 light chain 1-CKLO2 IgG1 SEQ ID NO

111 light chain 2-CKLO2 IgG1 SEQ ID NO

CKLO 2-SG 1095 (exemplary bispecific CLOK2 Crossmab, including SG1095Fc mutation)

112 heavy chain 1-CKLO 2-SG 1095 of SEQ ID NO:112

113 heavy chain 2-CKLO 2-SG 1095

114 light chain 1-CKLO 2-SG 1095

115 light chain 2-CKLO 2-SG 1095 SEQ ID NO

CKLO 2-SG 1099 (exemplary bispecific CKLO2 Crossmab, including SG1099Fc mutation)

116 heavy chain 1-CKLO 2-SG 1099 of SEQ ID NO

117 heavy chain 2-CKLO 2-SG 1099 of SEQ ID NO

118 light chain 1-CKLO 2-SG 1099

119 light chain 2-CKLO 2-SG 1099 of SEQ ID NO

CKLO 2-SG 1100 (exemplary bispecific CKLO2 Crossmab, including SG1100Fc mutation)

120 heavy chain 1-CKLO 2-SG 1100

121 heavy chain 2-CKLO 2-SG 1100

122 light chain 1-CKLO 2-SG 1100 of SEQ ID NO:122

123 light chain 2-CKLO 2-SG 1100 SEQ ID NO:123

CKLO 3-SG 1095 (exemplary bispecific CLOK3 Crossmab, including SG1095Fc mutation)

124 heavy chain 1-CKLO 3-SG 1095 of SEQ ID NO

125 heavy chain 2-CKLO 3-SG 1095

126 light chain 1-CKLO 3-SG 1095

127 light chain 2-CKLO 3-SG 1095 SEQ ID NO

CKLO 3-SG 1099 (exemplary bispecific CKLO3 Crossmab, including SG1099Fc mutation)

128 heavy chain 1-CKLO 3-SG 1099 of SEQ ID NO

129 heavy chain 2-CKLO 3-SG 1099 SEQ ID NO

130 light chain 1-CKLO 3-SG 1099 of SEQ ID NO

131 light chain 2-CKLO 3-SG 1099

CKLO 3-SG 1100 (exemplary bispecific CKLO3 Crossmab, including SG1100Fc mutation)

132 heavy chain 1-CKLO 3-SG 1100

133 heavy chain 2-CKLO 3-SG 1100

134 light chain 1-CKLO 3-SG 1100

135 light chain 2-CKLO 3-SG 1100 SEQ ID NO

Other anti-CCL 2 antigen-binding moieties:

136 heavy chain variable domain VH 2F2

137 light chain variable domain VL 2F2 SEQ ID NO

138 heavy chain variable domain VH murine 11K2(═ 11K2m)

Light chain variable domain of SEQ ID NO 139 VL murine 11K2(═ 11K2m)

140 heavy chain variable domain of SEQ ID NO: VH 1H11

141 light chain variable domain VL 1H11

Exemplary CCL2 and homologs (no signal peptide):

142 exemplary human CCL2(MCP1) -wild type (wt)

143 exemplary human CCL2(MCP1) -P8A variant of SEQ ID NO

144 exemplary human CCL2(MCP1) -T10C variant of SEQ ID NO

145 exemplary human CCL8(MCP2) -wild type (wt)

146 exemplary human CCL8(MCP2) -P8A variant of SEQ ID NO:146

SEQ ID NO:147 exemplary human CCL7(MCP3) -wild type (wt)

148 exemplary human CCL13(MCP4) -wild type (wt)

149 exemplary Macaca fascicularis CCL 2-wild type (wt)

SEQ ID NO:150 exemplary mouse CCL 2-wild type (wt)

Other exemplary constant heavy chain regions:

151GG 01-an exemplary human heavy chain constant region derived from IgG1, including mutations (Kabat EU numbering):

L234Y/P238D/T250V/V264I/T307P/A330K (suitable for increasing the affinity to human FcgRIIB and to other human FcgR);

-Q311R/P343R (adapted to increase the isoelectric point (pI) to enhance antigen uptake);

-N434A (adapted to increase affinity for FcRn to prolong the plasma half-life of the antibody); and

Q438R/S440E (suitable for inhibiting rheumatoid factor binding)

152GG 02-an exemplary human heavy chain constant region derived from IgG1, including mutations (Kabat EU numbering):

-M428L/N434A/Y436T (suitable for increasing the affinity to FcRn to prolong the plasma half-life of the antibody); and

Q438R/S440E (suitable for inhibiting rheumatoid factor binding)

153GG 03-exemplary human heavy chain constant region derived from IgG1 (comprising an IgG1 allotype sequence), including mutations (Kabat EU numbering):

Q438R/S440E (suitable for inhibiting rheumatoid factor binding)

154GG 04-human derived from IgG1 (comprising an IgG1 allotype sequence)

Heavy chain constant region, including mutations (Kabat EU numbering):

Q438R/S440E (suitable for inhibiting rheumatoid factor binding)

CKLO 2-GG 01 (exemplary bispecific CLOK2 Crossmab, including GG01 Fc mutation)

155 heavy chain of SEQ ID NO: 1-CKLO 2-GG 01

156 heavy chain 2-CKLO 2-GG 01 SEQ ID NO

157 light chain 1-CKLO 2-GG 01 SEQ ID NO

158 light chain 2-CKLO 2-GG 01

CKLO 2-GG 02 (exemplary bispecific CLOK2 Crossmab, including GG02 Fc mutation)

159 heavy chain 1-CKLO2 GG02 SEQ ID NO

160 heavy chain 2-CKLO2 GG02 SEQ ID NO

161 light chain 1-CKLO2 GG02 SEQ ID NO

162 light chain 2-CKLO2 GG02 SEQ ID NO

CKLO 2-GG 03 (exemplary bispecific CLOK2 Crossmab, including GG03 Fc mutation)

163 heavy chain 1-CKLO 2-GG 03 of SEQ ID NO:163

164 heavy chain 2-CKLO 2-GG 03 SEQ ID NO

165 light chain 1-CKLO 2-GG 03 SEQ ID NO

166 light chain 2-CKLO 2-GG 03 SEQ ID NO:166

CKLO 2-GG 04 (exemplary bispecific CKLO2 Crossmab, including GG04 Fc mutation)

167 heavy chain 1-CKLO 2-GG 04 SEQ ID NO

168 heavy chain 2-CKLO 2-GG 04 SEQ ID NO

169 light chain 1-CKLO 2-GG 04 SEQ ID NO

170 light chain 2-CKLO 2-GG 04 SEQ ID NO

CKLO 2-GG 03/GG04 (exemplary bispecific CKLO2 Crossmab, including GG03 Fc mutation in Knob (Knob) chain and GG04 Fc mutation in well (Hole) chain)

171 heavy chain 1-CKLO 2-GG 03/GG04 SEQ ID NO

172 heavy chain 2-CKLO 2-GG 03/GG04 SEQ ID NO

173 light chain 1-CKLO 2-GG 03/GG04 SEQ ID NO

174 light chain of SEQ ID NO 2-CKLO 2-GG 03/GG04

Designated monospecific unmodified anti-CCL 2 antibodies/antigens for anti-CCL 2 bispecific antibodies described herein Binding moieties

Bispecific anti-CCL 2 with unmodified VH/VL was designated as having mutations including L234A, L235A and P329G (PGLALA) IgG1 or IgG1 Crossmabs (see WO) 2016/016299)

Bispecific anti-CCL 2 antibodies	Alias name
		11K2//1G9-WT IgG1	CCL2-0049
11K2//1G9-PGLALA	CCL2-0043
		CNTO888//11K2-WT IgG1	CCL2-0048
CNTO888//11K2-PGLALA	CCL2-0042
		CNTO888//1G9-WT IgG1	CCL2-0051
CNTO888//1G9-PGLALA	CCL2-0045
		CNTO888//1A5-WT IgG1	CCL2-0050
CNTO888//1A5-PGLALA	CCL2-0044
		1A5//1G9-WT IgG1	CCL2-0052
1A5//1G9-PGLALA	CCL2-0046
		11K2//2F6-WT IgG1	CCL2-0056
11K2//2F6-PGLALA	CCL2-0053
		ABN912//11K2-WT IgG1	CCL2-0047
ABN912//11K2-PGLALA	CCL2-0041
		1A4//2F6-WT IgG1	CCL2-0057
1A4//2F6-PGLALA	CCL2-0054
		1A5//2F6-WT IgG1	CCL2-0058
1A5//2F6-PGLALA	CCL2-0055

Bispecific antibodies with modified VH/VL are designated as Crossmab (see WO) 2016/016299). According to Heavy chain constant domains (e.g., IgG1 wild-type, PGLALA, SG1095, SG1099, 1100) used with the addition of the suffix IgG1 Wild type, PGLALA, SG1095, SG1099, 1100

Example (b):

example A-1 monospecific anti-CCL 2 antibody

Production of monospecific anti-CCL 2 antibody and CCL2 antigen

Recombinant DNA technology

The DNA is manipulated using standard methods, such as those described in Sambrook, J.et al, Molecular cloning: A laboratory Manual; cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions.

Gene and oligonucleotide synthesis

In Geneart GmbH (Regensburg, Germany), the desired gene fragment was prepared by chemical synthesis. The synthesized gene fragment was cloned into an E.coli plasmid for propagation/amplification. The DNA sequence of the subcloned gene fragments was verified by DNA sequencing. Alternatively, short synthetic DNA fragments are assembled by annealing (annealing) chemically synthesized oligonucleotides or via PCR. The corresponding oligonucleotide primers were prepared by means of metabion GmbH (Planegg-Martinsried, Germany). Metabion GmbH (Planegg-Martinsried, Germany).

Description of basic/Standard mammalian expression plasmids

For expression of the desired gene/protein (e.g. full length antibody heavy chain, full length antibody light chain or CCL-2 molecule), a transcription unit is used comprising the following functional elements:

immediate early enhancer and promoter from human cytomegalovirus (P-CMV) including intron A,

-the human heavy chain immunoglobulin 5 'untranslated region (5' UTR),

a murine immunoglobulin heavy chain signal sequence,

the gene/protein to be expressed (e.g. full length antibody heavy chain or antibody light chain or CCL-2 molecule), and

-bovine growth hormone polyadenylation sequence (BGH pA).

In addition to the expression unit/cassette comprising the gene to be expressed, the basic/standard mammalian expression plasmid contains

An origin of replication from the vector pUC18 which allows replication of this plasmid in E.coli, and

a beta-lactamase gene, which confers ampicillin (ampicillin) resistance in e.

Generation of expression plasmids for recombinant monoclonal antibodies and CCL-2 molecules

The expression plasmid for transient expression of the monoclonal antibody and the CCL-2 antigen comprises, in addition to the respective expression cassettes, an origin of replication from the vector pUC18 (which allows the plasmid to replicate in e.coli), and a β -lactamase gene conferring ampicillin resistance to e.coli.

The corresponding immunoglobulin HC or LC or CCL-2 molecule transcription unit comprises the following functional elements:

-the human heavy chain immunoglobulin 5 'untranslated region (5' UTR),

a murine immunoglobulin heavy chain signal sequence, and

-bovine growth hormone polyadenylation sequence (BGH pA).

Antibodies 1A4, 1A5, 1G9, 2F6, CNTO888, murine and humanized 11K2, ABN912, respectively, are based on their VH And VL was generated as IgG1 wild-type and as IgG1 PGLALA/effector silencing Fc with kappa light chain

Transient expression and purification

Recombinant production was performed by transient transfection of HEK293 cells (derived from human embryonic kidney cell line 293) cultured in F17 medium (Invitrogen Corp.). To produce monoclonal antibodies, cells are co-transfected with plasmids containing the respective immunoglobulin heavy and light chains. For transfection, the "293-Fectin" transfection reagent (Invitrogen) was used. Transfection was performed according to the manufacturer's instructions. Cell culture supernatants were harvested three to seven (3-7) days after transfection. The supernatant is stored at reduced temperature (e.g., -80 ℃).

General information on recombinant expression of human immunoglobulins in, for example, HEK293 cells is provided in: meissner, P. et al, Biotechnol. Bioeng.75(2001) 197-203.

Antibodies were purified from cell culture supernatants by affinity chromatography using MabSelectSure-sepharose (GE Healthcare, Sweden) and Superdex200 size exclusion (GE Healthcare, Sweden) chromatography. Briefly, sterile filtered cell culture supernatants were captured on MabSelect SuRe resin equilibrated with PBS buffer (10mM Na2HPO4, 1mM KH2PO4, 137mM NaCl, and 2.7mM KCl, pH 7.4), washed with equilibration buffer and eluted with 25mM sodium citrate at pH 3.0. Eluted protein fractions were combined, neutralized with 2M Tris pH 9.0 and further purified by size exclusion chromatography using Superdex 20026/60 GL (GE Healthcare, Sweden) column equilibrated with 20mM histidine, 140mM NaCl, pH 6.0. Size exclusion chromatography fractions were analyzed by CE-SDS (Caliper Life Science, USA), and antibody-containing fractions were pooled and stored at-80 ℃.

Generation of recombinant CCL2

Wild-type CCL2 may be present as a monomer, but actually may form a dimer at physiological concentrations. This monomer-dimer equilibrium may be different and must be carefully considered in all in vitro experiments where different concentrations may be used. To avoid any uncertainty, we generated point mutation CCL2 variants: the P8A variant of CCL2 carries mutations in the dimerization interface, resulting in the inability to form dimers that result in a defined pure CCL2 monomer. In contrast, the T10C variant of CCL2 resulted in a fixed dimer of CCL2 (J Am Chem Soc.2013Mar 20; 135 (11): 4325-32).

The respective soluble CCL2 proteins (wild type, P8A or T10C variant) were purified from cell culture supernatants by cation exchange chromatography using SP-Sepharose HP (GE Healthcare, Sweden) and Superdex 200 size exclusion (GE Healthcare, Sweden) chromatography. Briefly, sterile filtered cell culture supernatant was diluted with 10mM KH2PO4, pH 5.0 to adjust conductivity <4 mS/cm. The diluted supernatant was loaded onto SP-Sepharose resin equilibrated at 10mM KH2PO4, pH 5.0, washed with equilibration buffer and eluted using a gradient to 10mM KH2PO4, 1M NaCl, pH 5.0. The eluted protein fractions were combined and further purified by size exclusion chromatography using a Superdex 20016/60 GL (GE Healthcare, Sweden) column equilibrated with 20mM histidine, 140mM NaCl, pH 6.0. Size exclusion chromatography fractions were analyzed by SDS-PAGE and analytical high performance size exclusion chromatography. The fractions containing CCL2 were combined and stored at-80 ℃.

Functional characterization (combination)

The T200 instrument was fitted with a Biacore Series S Sensor Chip CM 5. The system buffer was HBS-ET (10mM HEPES (pH 7.4), 150mM NaCl, 1mM EDTA, 0.05% (w/v) P20). The system was set to 37 ℃ and the sample buffer was system buffer for each measurement, additionally supplemented with 1mg/ml CMD (carboxymethyl dextran, Fluka).

An antibody capture system was established. 14000GARFc gamma (goat anti-rabbit Fc gamma) 111-005-046, Jackson ImmunoResearch) was immobilized at 25. mu.g/ml in 10mM sodium acetate buffer pH 5.0 at 25 ℃ by EDC/NHS coupling as described by the manufacturer. The capture system was regenerated by injection with HBS buffer (100mM HEPES pH 7.4, 1.5M NaCl, 0.05% (w/v) Tween 20) for 15 seconds at a rate of 20. mu.l/min, 10mM glycine buffer pH 2.0 for 1 minute, and then 10mM glycine buffer pH 2.25 for 1 minute in duplicate. In another example, murine monoclonal antibodies were captured on a biosensor by immobilizing 12700RU polyclonal rabbit anti-mouse (RAMIgG, GE Healthcare) antibody on a Biacore Series CM5 sensor as described above. The sensor was regenerated by injecting 10mM glycine buffer pH 1.7 for 3 minutes.

Antibody clone supernatants were diluted 1:2 in system buffer and captured at 5. mu.l/min for 1 min. After antibody capture, the system was washed with 2.5 fold concentrated system buffer at 80 μ l/min for 30 seconds, followed by 2 minutes of baseline stabilization. Analyte kinetics were performed at 30. mu.l/min. As analyte in solution, wt human CCL2 or monomeric CCL 2P 8A variant CCL2 was used. The analyte was injected at the highest concentration of 90 nM. The analyte contact time was 3 minutes and the dissociation time was 10 minutes, using Biaevaluation software v.3.0 according to the manufacturer GEHC instructions. The kinetic rates were clearly estimated using a 1:1 binding model with RMAX locality.

Antibodies and wild type (wt)Binding of human CCL2 and human CCL 2P 8A variants (monomers)

Summary pH-dependent CCL2 binding kinetics obtained from SPR analysis I

The T200 instrument was fitted with a Biacore Series S Sensor Chip CM 5. The system buffer was HBS-ET (10mM HEPES (pH 7.4), 150mM NaCl, 1mM EDTA, 0.05% (w/v) P20). In other embodiments, the pH of the system buffer is set to pH 8.3, pH 7.9, pH 7.4, pH 7.1, pH 6.7, pH 6.3, pH 5.9, pH 5.5. The system was set at 25 ℃ and the sample buffer was system buffer for each measurement, additionally supplemented with 1mg/ml CMD (carboxymethyl dextran, Fluka).

An antibody capture system was established. 13000MAb < h-Fc-pan > M-R10Z8E9-IgG (Roche) was fixed at 18g/ml in 10mM sodium acetate buffer pH 5.0 at 25 ℃ by EDC/NHS coupling as described by the manufacturer. The capture system was regenerated by injection of HBS buffer (100mM HEPES pH 7.4, 1.5M NaCl, 0.05% (w/v) Tween 20) at 20. mu.l/min, followed by 1 minute 15 seconds of injection with 10mM NaOH and two 1 minute injections of 10mM glycine buffer pH 2.5, each system buffer being injected with capture antibody diluted at 80nM concentration for 30 seconds at 10. mu.l/min. After antibody capture, the system was washed with 2.5 fold concentrated system buffer at 50 μ l/min for 30 seconds, followed by 2 minutes of baseline stabilization. The concentration-dependent analyte series was injected in 1:3 dilution steps, starting at 0nM (buffer control), 0.4nM, 1.1nM, and twice at 3.3nM, 30 nM. Analyte contact time was 3 minutes and dissociation time was 10 minutes, analyte kinetics were performed at 50 μ l/min.

The human antibody is captured as a ligand on the sensor surface:

human normal IgG as a positive control (H-N-IgG, Id.: 11717570, Roche),

anti-human CCL2 mAb (humanized 11k 2: CCL2-0002),

anti-human CCL2 mAb (AB912, CCL2-0003), and

Anti-human CCL2 mAb (CNTO888, CCL 2-0004);

system buffer as negative control.

Biaeevaluation software V.3.0 was used according to the manufacturer's GEHC instructions. Use of compounds having R _MAX The kinetic rates were determined by local 1:1 binding models.

Cross-reactive CCL homologs

Since CCL2(MCP-1) has high homology to CCL7(MCP-3), CCL8(MCP-2), CCL13(MCP-4), and these CCL chemokines are capable of binding to CCR2, the binding of anti-CCL 2 antibodies to these homologues was evaluated. The results are shown in FIG. 1. In addition to CNTO888(Mol Immunol.2012Jun; 51 (2): 227-33), which is described as selective for CCL2, other test antibodies all bound to CCL7 or CCL8 (showing cross-reactivity to CCL7 or CCL 8).

Biacore assay: anti-CCL 2 antibodies were evaluated for binding to CCL homologs, such as CCL2(MCP-1), CCL8(MCP-2), CCL7(MCP-3), and CCL13(MCP-4), at 25 ℃ using a Biacore T200 instrument (GE Healthcare). Mouse anti-human igg (fc) (GE Healthcare) was immobilized on each flow cell of the CM4 sensor chip using an amine coupling kit (GE Healthcare) according to the manufacturer's recommended setup. The antibody and analyte were diluted into ACES pH 7.4 buffer (20mM ACES,150mM NaCl,1mg/ml BSA, 0.05% Tween 20, 0.005% NaN 3). Antibodies were captured onto the anti-Fc sensor surface and recombinant human CCL homologous protein was injected onto the flow cell at 5nM and 20 nM. Wild-type CCL2(MCP-1), CCL8(MCP-2), CCL7(MCP-3) and CCL13(MCP-4) are commercially available from R & D Systems, while monomeric CCL2(P8A variant) is an antigen produced in the plant. The sensor surface was regenerated with 3M MgCl2 every cycle. Biacore T200 evaluation software version 2.0 (GE Healthcare) processing was used in conjunction with sensorgrams.

Functional characterization (biological)

CCR2 signaling I-calcium flux assay

THP-1 (human acute monocytic leukemia cell line; ATCC TIB-202) cells were cultured in RPMI 1640, 10% FBS, 1mM sodium pyruvate, 10mM HEPES, 50. mu.M beta-mercaptoethanol (supplier Thermo Fisher Scientific). On the day of assay, cell density was adjusted to 8.33X 10 in 25.8ml assay medium (RPMI 1640w/o FBS) ⁵ Individual cells/ml.Calcium Assay kits (FLIPR Calcium 4Assay Kit, Cat # R8142, Molecular Devices) were used to detect changes in intracellular Calcium in a homogeneous Assay format.

Dye loading solutions were prepared by mixing two vials of component A with 20ml of component B (HBSS buffer plus 20mM HEPES, pH 7.4) according to the manufacturer's Molecular Devices instructions. Mu.l of 1M Hepes (final assay concentration: 10mM) was added, followed by 516. mu.l of 250mM probenecid (final assay concentration: 2.5 mM). For the stock solution, 65.4mg probenecid (Sigma P8761) was dissolved in 465. mu.l 1N NaOH and 465. mu.l 1 XHBSS (Thermo Fisher Scientific) was added. 25.8ml of loading buffer is mixed with 25.8ml of assay medium having a volume sufficient for use in, for example, four microtiter plates (52.6 ml volume required; 10) ⁶ THP-1/ml). Transfer 120 μ l of cell suspension in loading buffer to each well of a black F-bottom 96 well cell culture plate. The mixture was incubated at room temperature for 3-4 hours.

Simultaneously, antibody and ligand solutions were prepared. Eight concentrations of each antibody have been tested from 30 μ g/ml to 0.025 μ g/ml (no serial dilutions, final concentration in wells). Each concentration was tested on two discs. All dilutions were made in assay medium as 10-fold concentrated solutions. As a reference antibody, human CCL2/JE/MCP-1 antibody (R & D Systems Cat # MAB279) was used. Ligand CCL2(R & D Systems Cat #279-MC-10) was prepared by dissolving 50. mu.g of CCL2 lyophilizate in 500. mu.l of RPMI 1640 (100. mu.g/ml) and transferring 400. mu.l to 10ml of assay medium (4. mu.g/ml stock solution). As a stimulation control, ionomycin (ionomycin) (Sigma Cat # I-0634) (1 mg ionomycin was dissolved in 1340. mu.l DMSO (Sigma Cat # D-8779), 1mM) was used. Mu.l of 1mM stock solution was diluted in 1990. mu.l of assay medium (5. mu.M, final assay concentration 500 nM). 100 μ l were pipetted into corresponding control wells of polypropylene MTP.

Antibody dilutions and CCL2 were preincubated in two V-shaped polypropylene 96-well plates. 50 μ l of 4 μ g/ml stock solution CCL2 (final 400ng/ml CCL2) and 50 μ l of 10-fold concentrated antibody dilution were pipetted into the wells. The plates were incubated at room temperature for 30-60 minutes.

After incubation, the cell plates and compound plates were transferred directly to3(Molecular Devices) and calcium determination (excitation 485nm, emission 525nm) as described in the system manual. The read interval is a few seconds.

Results

Table 1:40ng/ml PMA and 4. mu.M ionomycin were used as positive controls. The table includes EC where n is 2 ₅₀ Average value.

anti-CCL 2 antibodies inhibit the ability of CCL2, which is a CCR2 receptor expressed on monocytes, to induce internalization

To prevent ligand-induced internalization of CCR2 on myeloid cells, we established an in vitro assay and characterized anti-CCL 2 antibodies. Monocytes were isolated from peripheral blood of healthy donors by magnetic separation using a commercial kit (Stemcell, catalog # 15068). To block Fc Rs, monocytes were incubated with normal human IgG (Privigen, CSL Behring) at a final concentration of 500. mu.g/ml in FACS buffer (PBS + 0.2% BSA) on ice for 50 min. Then the cells are detachedThe heart was washed once more with FACS buffer for 10 min (300Xg, 4 ℃) and stored on ice. Dilutions of anti-CCL 2 antibody (50. mu.l each) were prepared in 96U-bottomed wells (BD) (performed simultaneously at 4 ℃ and 37 ℃). The monocytes were split and resuspended in culture medium (RPMI 1640; 10% FCS; 2mM L-glutamine) and incubated at 4 ℃ and 37 ℃ respectively until further use. Recombinant CCL2 (50. mu.l; final concentration 100ng/ml) was added to the prepared antibody dilution (variable concentration) at 4 ℃ and 37 ℃ and 100. mu.l of the monocyte suspension (2X 10) ⁵ Individual cells/well) was added to a total volume of 200 μ l of CCL 2/anti-CCL 2 mixture and the cells were incubated at 4 ℃ and 37 ℃ for 1 hour 30 minutes and then centrifuged at 300xg, 4 ℃. From now on, all steps were performed using pre-cooled buffer: cells were washed with 250. mu.l FACS buffer and, in addition, stained for the CCR2 receptor (using a commercially available CCR2-APC conjugate or the appropriate ctrl-APC of the same type according to standard FACS protocols: aliquots were stained 5. mu.l/10 ⁶ Cells harbored CD192(CCR2) APC (BioLegend, #357208, clone K036CZ/mIgG2a), and appropriate isotype ctrl antibodies: 20 μ l/10 ⁶ Cell mIgG2a k APC BD Biosciences, #400222, clone MOPC-173).

Receptor expression was then analyzed on a FACS Canto II and CCR2 internalization was calculated as follows:

no internalization: cells were analyzed without ligand (rec. ccll 2) incubation.

100% internalization: the expression level of CCR2 on cells previously incubated with rec was minimized. CCL2

Inhibition of CCL 2-mediated chemotaxis on human THP-1 cells

CCR2 ⁺ Migration of THP1 cells to the CCL2 gradient was tested as follows. Mononuclear THP1 cells (TIB-202 ^TM ) Cultured in RPM11640 medium (PAN, Cat # P04-17500), which was supplemented with FCS and L-glutamine. Cells are typically subcultured 2 to 3 times and then used again for migration assays, and then starved overnight in media with reduced FSC content (1.5% instead of 10% FCS). Cells were counted and incubated with 10. mu.g/ml normal human IgG (Invitrogen, Cat # 12000; blocking FcgR) for 15 min at room temperature.

At the same time, anti-CCL 2 antibody (and/or control) was added to the lower chamber of an HTS Transwell 96-well disc system (Corning, Cat # 3386; pore size 3 μm) containing serum-free medium and 25ng/ml rhCCL-2 (R)&D Systems, catalog # 279-MC). Insert plates were then attached to the lower chamber plate and 75 μ l (1.5 × 10) with or without 5 μ g/ml antibody/isotype was added to each insert ⁵ Cells) of the above cell suspension (including IgG-blockers). The plates were covered and incubated overnight at 37 ℃ in a CO2 incubator (5% CO 2).

The insert disk was removed and Cell-titer-glo substrate (Promega, catalog # G758) was added to each well of the lower chamber disk to measure the viability of the migrating cells. After 1 hour incubation on a shaker at 300rpm (sealed cover plate), 200. mu.l of each well was transferred to a Microfluor black 96-well plate (VWR, Cat # 735-. Fold change was calculated as the ratio of the number of migrating cells (Cell Titer Glo, RLU) with IgG control antibody to anti-CCL 2 antibody. The results of 5-10 replicates of each condition are shown in table 2 below:

TABLE 2

Assessment of clearance of human CCL2 immune complexes with a monospecific (single paratope) anti-CCL 2 antibody in mice

To assess the ability of the single paratope antibody to form an immune complex with wild-type human CCL2, a preformed immune complex consisting of anti-CCL 2 single paratope antibody (20mg/kg) and wild-type human CCL2(0.1mg/kg) was administered at 10ml/kgSingle dose administration of human FcRn transgenic mice via the tail vein (B6. Cg-Fcgrt) ^tm1Dcr Tg (FCGRT)32Dcr/DcrJ, Jackson Laboratory). Blood was collected at 5 minutes, 7 hours, 1 day, 2 days, 3 days and 7 days after administration. Serum was prepared by immediately centrifuging blood at 14,000rpm for 10 minutes at 4 ℃ and storing the serum at-80 ℃ or below-80 ℃ until measured. Table 3 below lists the single paratope antibodies tested. Antibodies with SG1 Fc had similar fey receptor binding as wild type, while antibodies with SG105 Fc were Fc γ receptor binding silent.

The effect of immune complex clearance of each anti-CCL 2 monoparate antibody on hCCL2 clearance in vivo was assessed by comparing anti-CCL 2 antibody with fcy receptor binding (SG1 ═ IgG1 wild type with intact fcy receptor binding; solid line) to anti-CCL 2 antibody with fey receptor binding silencing (SG105 ═ IgG1 without fcy receptor binding; dotted line), as shown in figure 2. Figures 2a to 2g each show hCCL2 serum concentrations over time following injection of pre-formed immunocomplexes consisting of hCCL2 and the respective anti-CCL 2 antibodies into FcRn transgenic mice, (with two different Fc portions: SG1 ═ IgG1 wild type with intact fey receptor binding, and SG105 ═ IgG1 with no fey receptor binding). Antibody curves were analyzed by non-compartmental analysis using Phoenix 64 (Pharsight/Certara). AUCinf is estimated by extrapolation to infinity using the linear logarithmic trapezoidal rule. Clearance values are defined as dose/AUCinf. This difference in clearance, also expressed as fold change, was calculated by dividing hCCL2 clearance of antibodies with Fc γ receptor binding (SG1) by hCCL2 clearance of antibodies with Fc γ receptor binding silencing (SG105) (table 3 below). The data in table 3 below show that for all tested single paratope antibodies, clearance of human CCL2 by Fc γ receptor binding antibody (SG1 ═ IgG1 wild type with intact Fc γ receptor binding) was similar to clearance of human CCL2 by Fc γ receptor binding silencing antibody (SG105, no Fc γ receptor binding). This indicates that immune complex-mediated CCL2 clearance by the tested single paratope antibodies was ineffective.

Table 3: pre-formed administration of anti-CCL 2 monospecific antibody (20mg/kg) and wild type human CCL2(0.1mg/kg) Clearance values of wild-type CCL2 following immunocomplex (IgG1 wild-type (SG1) or IgG1 Fc receptor silencing (SG105)

Measurement of total human CCL2 concentration in serum by Electrochemiluminescence (ECL)

The concentration of total human CCL2 in mouse serum was measured by ECL. 3ug/mL of anti-CCL 2 antibody (F7(Biolegend) or clone MAB679(R & D Systems)) was immobilized on a Multi-ARRAY 96 well plate (Meso Scale Discovery) overnight and then incubated in blocking buffer at 30 ℃ for 2 hours. anti-CCL 2 MAB679 was used as a capture antibody for samples containing humanized 11K2, 1a4, or 1a5 antibodies. anti-CCL 2 clone 5D3-F7 was used for samples comprising ABN912, CNTO888, 1G9, 2F6H antibodies. Human CCL2 calibration curve samples, quality control samples, and mouse serum samples were prepared by dilution in dilution buffer and incubation with excess drug for 30 minutes at 37 ℃. Thereafter, the sample was added to the tray on which CCL2 was immobilized and bound at 30 ℃ for 1 hour, followed by washing. Thereafter, SULFO TAG NHS ester (Meso Scale Discovery) -labeled anti-human Fc (clone: JDC-10, southern Biotech) was added and the discs were incubated at 30 ℃ for 1 hour, followed by washing. Read Buffer T (x4) (Meso Scale Discovery) was immediately added to the disc and the signal was detected with SECTOR Imager 2400(Meso Scale Discovery). Human CCL2 concentration was calculated based on the response of the calibration curve using analytical software SOFTmax PRO (Molecular Devices).

Measurement of the concentration of anti-CCL 2 antibody in serum by enzyme-linked immunosorbent assay (ELISA)

The concentration of anti-CCL 2 antibody in mouse serum was measured by ELISA. Anti-human IgG kappa chains (antibody solution) were dispensed onto Nunc MaxiSorp disks (thermoldisser) and allowed to stand overnight at 4 ℃ to prepare anti-human IgG immobilized disks. Calibration curves and samples were prepared with 1% pooled mouse serum. Then, the sample was dispensed onto a disk fixed with anti-human IgG and allowed to stand at 30 ℃ for 1 hour. Subsequently, goat anti-human IgG (gamma-chain specific) with HRP conjugate (Southern Biotech) was added and reacted at 30 ℃ for 1 hour. The color reaction was carried out using TMB substrate (Life Technologies) as a substrate. After the reaction was terminated with 1N sulfuric acid (Wako), the absorbance at 450nm was measured by a microplate reader. The concentration in the mouse plasma was calculated from the absorbance of the calibration curve using the analytical software SOFTmax PRO (Molecular Devices).

Assessment of clearance of endogenous mouse CCL2 immune complexes in mice with a single paratope antibody

In addition to the above results, which indicate that immune complex-mediated CCL2 clearance by the tested single paratope antibodies was ineffective, further evaluations were performed.

To evaluate the ability of the single paratope antibody to form and clear immune complexes with endogenous mouse CCL2, mice were administered with a cross-reactive 11K2 anti-CCL 2 single paratope antibody. Humanized 11K2H2-SG1(IgG1 wild-type ═ fcgamma receptor binding) or humanized 11K2-SG105 (fcgamma receptor binding silent) antibody was administered intravenously at a single dose of 20mg/kg, and Balb/c mice were administered via the tail vein at a single dose of 10 ml/kg. Blood was collected before administration, 5 minutes, 7 hours, 1 day, 2 days, 3 days, and 7 days after administration. Serum was prepared by immediately centrifuging blood at 14,000rpm for 10 minutes at 4 ℃ and storing the serum at-80 ℃ or below-80 ℃ until measured.

FIG. 3 shows the time course of serum total mouse CCL2 concentration of humanized 11K2-SG1 and 11K2-SG105 in mice, as well as antibody-time curves.

As shown in fig. 3, there was no difference in the level of accumulation of mouse CCL2 between 11K2-SG105 (fey receptor binding silent Fc) and 11K2-SG1(IgG1 wild-type ═ fey receptor binding Fc). This indicates that the injected antibodies have no or very little Fc γ receptor mediated clearance of endogenous mouse CCL 2. Via multimeric conjugation of Fc γ receptors, the antigen in the immune complex cleared faster than uncomplexed antigen, indicating that the 11K2 antibody failed to form an immune complex with endogenous mouse CCL 2.

Enzyme-linked immunosorbent assay (ELISA) measurement of mouse CCL2 concentration in mouse serum

The concentration of mouse CCL2 in mouse serum was measured by adapting reagents from a commercially available mouse CCL2 ELISA kit (R & D Systems). In addition to preparing the calibration curve samples, the manufacturer's protocol was followed. Purified recombinant mouse CCL2 was substituted as a standard to replace the manufacturer's protein. For samples collected after antibody injection, calibration curve samples and samples were prepared by injecting the antibody in 2.5% mouse serum at a concentration of 40. mu.g/ml and incubated at 37 ℃ for 30 minutes. Subsequently, samples were dispensed into anti-human CCL2 fixed disks and incubated at 30 ℃ for 2 hours. By adding mouse MCP-1 conjugate and incubating at 30 ℃ for 2 hours, then adding substrate and stopping the solution.

For samples taken prior to antibody injection, Mouse MCP-1Ultra-Sensitive Kit (Meso Scale Discovery) was used according to the manufacturer's instructions. No antibody was incorporated into the sample prior to addition to the dish.

The concentration of anti-CCL 2 antibody in mouse serum was measured by ELISA. Anti-human IgG kappa chains (antibody solution) were dispensed onto Nunc MaxiSorp disks (thermoldisser) and allowed to stand overnight at 4 ℃ to prepare anti-human IgG immobilized disks. Calibration curves and samples were prepared with 1% pooled mouse serum. Then, the sample was dispensed onto an anti-human IgG-immobilized plate and allowed to stand at room temperature for 1 hour. Subsequently, a mouse anti-human IgG HRP (clone JDC-10, Southern Biotech) was added and the reaction was carried out at room temperature for 30 minutes. The color reaction was performed using ABTS substrate (KPL) as a substrate, and the absorbance at 405nm was measured by a microplate reader. Concentration in mouse plasma was calculated from absorbance of calibration curve using analytical software SOFTMax PRO (Molecular Devices)

Conclusion of different mouse PK Studies with monospecific (single paratope) anti-CCL 2 antibodies

Summarizing the results of the mouse PK study, indeed no tested single paratope antibody was shown to effectively clear CCL2 from circulation. These data indicate that the single paratope antibody is unable to form an immune complex with CCL2 and is unable to efficiently clear it from circulation.

In contrast, a bispecific anti-CCL 2 antibody with two different antigen binding moieties/sites (biparatopic anti-CCL 2 antibody) was effective to form an immune complex with CCL2 and clear it from the circulation, as described below.

Example B-1

Bispecific (bispecific) anti-CCL 2 antibodies

Generation of several antibodies with two different antigen-binding moieties (each binding to another) that bind to two different specific epitopes on human CCL2 Complement) bispecific anti-CCL 2 antibodies

Brief introduction to the drawings

To test whether single binding or cross-linking of antigens has a significant effect on CCL2 clearance in vivo, we used bispecific CrossMab Technology (see, e.g., WO 2009/080252, WO 2015/150447), WO 2009/080253, WO 2009/080251, WO 2016/016299, Schaefer et al, PNAS,108(2011)1187-1191 and Klein at, MAbs 8(2016)1010-20) to generate bispecific anti-CCL 2 antibodies (bispecific (═ biparatopic) CrossMabs) with 2 different antigen binding moieties/sites that bind to 2 different epitopes of CCL 2. These molecules were first characterized in vitro for their biochemical and functional properties, but in mouse coinjection studies they were also used as a tool for the assessment of CCL2 clearance in vivo. To assess the clearance potential for Fc γ receptor (FcgR) binding-based mediated clearance (e.g., in Igawa et al, Immunological Reviews 270(2016) 132-151, WO2012/122011, and WO2016/098357, and WO 2013/081143), we generated all crosssmabs, which are wild-type huIgG1 bound to FcgR and having a modified human IgG1 constant chain, that have reduced/eliminated binding to FcgR effector silencing molecules (e.g., IgG1 with mutations L234A, L235A, P329G (Kabat EU numbering).

Identification of appropriate anti-CCL 2 antibody pairs-biparatopic antibody arms were selected by sandwich ELISA.

A sandwich ELISA was performed to identify antibody pairs that did not compete for binding to human CCL 2. 384 well maxisorp (nunc) discs were coated with 1 μ g/mL of 7 indicated capture antibodies (Arm 1) and blocked with 2% BSA. Biotinylation (NHS-PEO) ₄ WT human CCL2(20ng/mL) from Biotin, Pierce) was incubated with an excess of the same 7 antibodies (Arm 2) (1. mu.g/mL) or blocking buffer for 1 hour at 37 ℃. After incubation, the mixture was added to blocked ELISA plates and incubated at room temperature for 1 hour. Detection of disc-bound CCL2 was performed using streptavidin (streptavidin) HRP followed by TMB One Component substrate (Lifetech). The generation of the signal was stopped by 1N HCl acid (Wako). For each capture antibody, the o.d. of wells without competing antibody was set to 100% signal. The o.d. of blank wells without CCL2 added was set to 0% signal. Nine pairs of antibodies that did not show strong competition for CCL2 binding in both directions were selected as candidates for generating bispecific crosssmab antibodies.

Generation and characterization of biparatopic anti-CCL 2 antibodies and immune complexes

Production of biparatopic anti-CCL 2 antibodies in bispecific CrossMab Format recombinant DNA technology

The DNA is manipulated using standard methods, as described in Sambrook, J.et al, Molecular cloning: A laboratory Manual; cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions.

Gene and oligonucleotide synthesis

Description of basic/Standard mammalian expression plasmids

For expression of the desired gene/protein (e.g. antibody heavy chain or antibody light chain), a transcription unit is used comprising the following functional elements:

the immediate early enhancer and promoter from human cytomegalovirus (P-CMV) including intron A,

the human heavy chain immunoglobulin 5 'untranslated region (5' UTR),

A murine immunoglobulin heavy chain signal sequence,

the gene/protein to be expressed (e.g.full length antibody heavy chain or MHC class I molecule), and

bovine growth hormone polyadenylation sequence (BGH pA).

The basic/standard mammalian expression plasmid contains, in addition to the expression unit/cassette comprising the gene to be expressed

An origin of replication from the vector pUC18 which allows the plasmid to replicate in E.coli, and

beta-lactamase gene, which confers ampicillin (ampicillin) resistance in E.coli.

Generation of expression plasmids for recombinant monoclonal antibodies

The recombinant monoclonal antibody genes encode the respective immunoglobulin heavy and light chains.

In addition to the immunoglobulin heavy or light chain expression cassette, the expression plasmid used for transient expression of the monoclonal antibody molecule also contains an origin of replication from the vector pUC18 that allows replication of the plasmid in e.coli, and a beta-lactamase gene that confers ampicillin resistance in e.coli.

The transcription unit of the respective antibody heavy or light chain comprises the following functional elements:

the human heavy chain immunoglobulin 5 'untranslated region (5' UTR),

A murine immunoglobulin heavy chain signal sequence,

respective antibody heavy or light chain cDNA sequences and

bovine growth hormone polyadenylation sequence (BGH pA).

Transient expression and analytical characterization

General information on recombinant expression of human immunoglobulins in, for example, HEK293 cells is provided in: meissner, P. et al, Biotechnol. Bioeng.75(2001) 197-203. To generate the following bispecific antibodies, the CrossMab technique described in WO 2016/016299 was used, in which VH/VL had been exchanged in one antibody arm, while the CH1/CL interface of the other antibody arm had been modified by charge modification, in combination with the knob-into-holes (knobs-int-holes) technique in the CH3/CH3 interface to promote heterodimerization. Exemplary sequences for all four antibody chains applying this technique are provided for CNTO888//11K2-WT IgG1 (see SEQ ID NO: 104 through SEQ ID NO: 107)

List of bispecific (biparatopic) anti-CCL 2 crosssmab antibodies generated with wild-type IgG1(WT IgG1) (wild-type IgG1 indicates no modifications/mutations that would affect Fc receptor binding, however including heterodimerization techniques such as knob (knob) into hole (hole))

List of bispecific (biparatopic) anti-CCL 2 crosssmab antibodies with IgG1 including Fc γ receptor silent mutations L234A, L235A, P329G (Kabat EU numbering) (IgG1-PGLALA)

Purification of biparatopic anti-CCL 2 antibody

The biparatopic anti-CCL 2 antibody containing cell culture supernatant was filtered and purified by up to three chromatography steps. An ion exchange chromatography step is optionally performed between the capture and polishing steps, depending on the purity of the eluent from the capture step.

Biparatopic anti-CCL 2 antibodies were purified from cell culture supernatants by affinity chromatography using MabSelectSure-sepharose (GE Healthcare, Sweden), POROS 50hs (thermofisher scientific), and Superdex 200 size exclusion (GE Healthcare, Sweden) chromatography. Briefly, sterile filtered cell culture supernatants were captured on MabSelect SuRe resin equilibrated with PBS buffer (10mM Na2HPO4, 1mM KH2PO4, 137mM NaCl, and 2.7mM KCl, pH 7.4), washed with equilibration buffer and eluted with 25mM sodium citrate at pH 3.0. Eluted protein fractions were pooled and neutralized with 2M Tris, pH 9.0.

Ion exchange chromatography is an optional second purification step using POROS 50hs (thermolither scientific), equilibrated and washed with 20mM histidine pH 5.6 and loaded with diluted capture step eluent, gradient chromatography with 20mM histidine, 0.5M NaCl at pH 5.6, ion exchange chromatography fractions analyzed with CE-SDS LabChip GX II (PerkinElmer) and pooled fractions containing Crossmab.

Size exclusion chromatography on Superdex 200(GE Healthcare) was used as the second or third purification step. Size exclusion chromatography was performed in 20mM histidine buffer, 0.14M NaCl, pH 6.0. Size exclusion chromatography fractions were analyzed by CE-SDS LabChip GX II (Perkinelmer) and the crosssmab containing fractions were pooled and stored at-80 ℃.

Satisfactory product quality was obtained in the case of replacement of POROS 50hs (thermofisher scientific) size exclusion chromatography on Superdex 200(GE Healthcare) with desalting chromatography on HiPrep 26/10 desaling (GE Healthcare) in 20mM histidine buffer, 0.14M NaCl, pH 6.0.

The protein concentration of the antibody preparation was determined by measuring the Optical Density (OD) at 280nm using the molar extinction coefficient calculated based on the amino acid sequence.

The purity and integrity of the antibodies were analyzed by CE-SDS using LabChip GX II (PerkinElmer) with the Protein Express Chip and HT Protein Express Kit. Using biosoite High Resolution SEC,a5 μm analytical size exclusion column (Waters GmbH) was used to determine the total content of the antibody preparation by high performance SEC using 200mM K2HPO4/KH2PO4, 250mM KCl, pH 7.0 as running buffer. When analyzed by CE-SDS, the average purity was between 94-100%, and the monomer content>95％(SEC)。

Functional characteristics of bispecific (biparatopic) anti-CCL 2 antibodies

Affinity measurement (binding)

Approximately 1200 Resonance Units (RU) of the capture system (20. mu.g/ml goat anti-human IgG Fc; order number: 109. sup. 005. sup. sub. 098; Jackson Immuno Research) were coupled to the C1 chip (GE Healthcare BR-1005-35) at pH 5.0 by using the amine coupling kit provided by GE Healthcare. The sample and system buffer was PBS-T (10 mM phosphate buffered saline including 0.05% Tween 20), pH 7.4. The flow cell was set at 25 ℃ and the sample block at 12 ℃ and perfused twice with running buffer. Bispecific antibodies were captured by injecting 2. mu.g/ml solution at a flow rate of 10. mu.l/min for 60 seconds. Measurement by injection of human CCL2(wt) in solutions of different concentrations for 150 seconds starting from a 1:10 dilution of 30nM at a flow rate of 30. mu.l/min The amount is associated. The dissociation phase was monitored for 1200 seconds and triggered by switching from the sample solution to the running buffer. By adding 0.85% H ₃ PO ₄ The solution was washed at a flow rate of 10. mu.l/min for 60 seconds to regenerate the surface. The overall refractive index difference (Bulk reactive index difference) was corrected by subtracting the response obtained from goat anti-human IgG Fc surface. Blank injections were also subtracted (double reference). For the calculation of kinetic parameters, the Langmuir 1:1 model was used.

Formation of a natural immune complex in the presence of a wild-type antigen.

All protein samples (bispecific anti-CCL 2CrossMab antibody and antigen) were rebuffered in 1x PBS, pH 7.4 using either dialysis or centrifugal ultrafiltration devices.

Cross Mab sample dilution series from 2.0 to 0.1mg/mL were prepared. Likewise, the concentration of the antigen solution in PBS ranges from 0.012 to 0.23 mg/mL. The concentration was chosen to allow mixing of equal volumes to achieve a constant molar ratio of 1:1 (antibody: CCL2 complex). The following antigens were used in this study: wild-type CCL 2.

Equal volumes of the pre-diluted CrossMab and CCL2 formulations were mixed and incubated at 37 ℃ for 1 hour, then the samples were applied to a Superose6(GE Healthcare #2039) column, pre-equilibrated with PBS, and eluted at a flow rate of 0.5 mL/min. A total of 100. mu.g or the maximum possible volume of 250. mu.L was applied and antibody and antigen alone were used as controls.

SEC-MALLS data were recorded using an OptiLab rEX refractive index detector and a miniDAWN Treos MALLS detector (both from Wyatt Inc.). The SEC-MALLS signal was processed using Astra V5 software (Wyatt).

Illustration of the drawings

A large number of polymers/oligomers

+ + Medium number of polymers/oligomers

+ Small amount of Polymer/oligomer

0 is only dimer or less

CCR2 reporter gene assay to study neutralization characteristics of anti-CCL 2 antibodies

Tango ^TM CCR2-bla U2OS cells were purchased from Invitrogen, Germany to study the effect of CCL2 neutralizing antibody constructs. These reporter cells contain the human chemokine (C-C Motif (Motif)) receptor 2(CCR2) linked to the TEV protease site and stably integrated into Tango ^TM Gal4-VP16 transcription factor in GPCR-bla U2OS parental cell line. The parent cell line stably expresses beta-rhodopsin (arrestin)/TEV protease fusion protein and beta-lactamase (bla) reporter gene under the control of UAS response element. The addition of the natural ligand MCP1 ═ CCL2 resulted in an indication of reporter gene activity, which can be measured by cleavage of FRET-functional substrates.

In principle, the assays and cell processing procedures were performed according to the provider manual. Briefly, CCR2-U2OS cells were seeded at a density of 2X104 cells/well (96er black transparent bottom plate, Cat #655090, Greiner Bio-one) in 50. mu.l assay Medium (Freestyle 293Expression Medium, Cat #12338-018, Invitrogen). Simultaneously, serial dilutions of different test antibody and CCL 2-antigen solutions were prepared at c ═ 4x final concentration. Then, a CCL 2-antigen/antibody mixture was prepared and preincubated at room temperature for 2 to 3 hours (hrs). 50 μ l of the CCL 2/antibody solution indicated was transferred to U2OS cells expressing CCR2 and incubated for 18 hours at 37 ℃ in a humidified incubator with 5% CO 2. Only assay medium was used as control.

The next day, CCF4 substrate (catalog # K1089, Invitrogen) was prepared as a beta-lactamase loading solution (catalog # K1085, Invitrogen) and added to the cells at 20 μ l/well. The substrate solution was incubated for 2 hours at room temperature in the dark.

Finally, the fluorescence wavelength was determined using a Spectra Max (M4) reader (Molecular devices) at the following wavelengths (Ex/Em 409nm/460nm blue; Ex/Em 409nm/530nm green) and according to the following formula: the ratio (sample-blue x-control-blue x)/(sample-green x-control-green x) was calculated as the blue/green fluorescence ratio after subtraction of assay medium control.

After pH engineering, we characterized their ability to inhibit CCL 2-induced CCR2 signaling against the final LO candidate (CKLO 1-4). In this case, the neutralization properties were evaluated only by the monomeric variant of rec. CCL2 protein at a final concentration of about 15 ng/ml.

Assessment of clearance of human CCL2 immune complexes with biparatopic antibodies in mice

To assess the ability of the biparatopic antibody to form an immune complex with wild-type human CCL2, Balb/c mice were administered tail vein with a single dose of 10ml/kg of a preformed immune complex consisting of anti-CCL 2 biparatopic antibody (20mg/kg) and wild-type human CCL2(0.1 mg/kg). Blood was collected 5 minutes, 7 hours, 1 day, 3 days and 7 days after administration. Serum was prepared by immediately centrifuging blood at 14,000rpm for 10 minutes at 4 ℃ and storing the serum at-80 ℃ or below-80 ℃ until measured. The tested biparatopic bits are listed in table 4 below. Antibodies with WT IgG1 Fc have similar Fc γ receptor binding as wild type, while antibodies with PGLALA Fc are silent for Fc γ receptor binding. The results are shown in FIGS. 4a to 4 i.

The effect of immune complex clearance of various anti-CCL 2 biparatopic antibodies on hCCL2 clearance in vivo was assessed by comparing anti-CCL 2 antibodies with fcy receptor binding (solid line) to anti-CCL 2 antibodies with fcy receptor silencing (PGLALA, dashed line), as shown in figures 4a-4 i. Antibody curves were analyzed by non-compartmental analysis using Phoenix 64 (Pharsight/Certara). AUCinf is estimated by extrapolation to infinity using the linear logarithmic trapezoidal rule. Clearance values are defined as dose/AUCinf. This difference in clearance, also expressed as fold change, was calculated by dividing hCCL2 clearance of antibodies with Fc γ receptor binding (SG1) by hCCL2 clearance of antibodies with Fc γ receptor binding silencing (PGLALA) (table 4 below). The data in table 4 below show that the clearance of Fc γ receptor (FcgR) binding antibody (WT IgG1) to human CCL2 is superior to that of Fc γ receptor binding silencing antibody (PGLALA, with IgG1 Fc domain (abat EU numbering) containing mutations L234A, L235A, P329G). This indicates that immune complex-mediated clearance of CCL2 by the tested biparatopic antibodies was more effective. In addition, several biparatopic antibodies showed a large fold change in clearance values, such as CNTO// humanized 11K2(CNTO//11K 2).

Fold change was calculated by dividing hCCL2 clearance of the antibody with WT Fc γ r (fcgr) binding by hCCL2 clearance of the antibody with PGLALA. As shown in fig. 4a-4i and table 4 below, CNTO//11k2 showed a maximum of 21.5 fold change between antibodies with FcgR-bound IgG1 wild-type (WT) and FcgR-bound silent antibodies (PGLALA). This indicates that immune complex-mediated clearance by CNTO//11k2-WT IgG1 is most effective in all variants.

Table 4: clearance values for wild-type CCL2 following preformed immunocomplexes of anti-CCL 2 biparatopic antibody (20mg/kg) and wild-type human CCL2(0.1mg/kg)

The concentration of total human CCL2 in mouse serum was measured by ECL. anti-CCL 2 antibody 2F2-SG1 at 3ug/mL was immobilized on a Multi-ARRAY 96 well plate (Meso Scale Discovery) overnight and then incubated in blocking buffer for 2 hours at 30 ℃. Human CCL2 calibration curve samples, quality control samples, and diluted mouse serum samples were incubated with denaturing buffer consisting of 9% SDS at 37 ℃ for 30 minutes, or with denaturing buffer consisting of glycine HCl buffer pH2.0-2.5 at 37 ℃ for 10 minutes. The purpose of the denaturation buffer was to dissociate human CCL2 from the biparatopic antibody. Thereafter, the samples were diluted 10-fold and added to the plates fixed with anti-CCL 2 and bound at 30 ℃ for 1 hour, followed by washing. Then, the SULFO TAG-labeled MCP-1 antibody was added, and the disc was incubated at 30 ℃ for 1 hour, followed by washing. Read Buffer T (x4) (Meso Scale Discovery) was immediately added to the disc and the signal was detected with SECTOR Imager 2400(Meso Scale Discovery). Human CCL2 concentration was calculated based on the response of the calibration curve using analytical software SOFTmax PRO (Molecular Devices).

The concentration of anti-CCL 2 antibody in mouse serum was measured by ELISA. Anti-human IgG kappa chains (antibody solution) were dispensed onto Nunc MaxiSorp disks (thermoldisser) and allowed to stand overnight at 4 ℃ to prepare anti-human IgG immobilized disks. Calibration curves and samples were prepared with 1% pooled mouse serum. Then, the sample was dispensed onto a disk fixed with anti-human IgG and left to stand at 30 ℃ for 1 hour. Subsequently, goat anti-human IgG (gamma-chain specific) with HRP conjugate was added (Southern Biotech) and reacted at 30 ℃ for 1 hour. The color reaction was performed using ABTS substrate (KPL) as a substrate, and the absorbance at 450nm was measured by a microplate reader. The concentration in the mouse plasma was calculated from the absorbance of the calibration curve using the analytical software SOFTmax PRO (Molecular Devices).

Summary of the study

Summarizing the data from the mouse PK study, the tested biparatopic antibodies were more effective in clearing human CCL2 than the same dose of single paratopic antibody. For the single paratope antibodies, there was little difference in antigen clearance between antibodies with silencing of WT FcgR binding versus FcgR binding (table 3). In contrast, there was a large difference in antigen clearance between biparatopic antibodies with silencing of WT FcgR binding and FcgR binding (table 4), indicating that the biparatopic antibodies tested were effective in clearing human CCL 2. The combination of CNTO888//11K2 was chosen for further antibody engineering as it shows the greatest chance on clearance.

Example B-2 anti-CCL 2 antibody with modified variable Domain and CDR (ion-dependent/pH-dependent binding) Body

The modification results in ion-dependent/pH-dependent binding

To generate pH-dependent anti-CCL 2 antibodies, all CDRs of mAb CNTO888 and humanized 11K2 were subjected to histidine scan induced mutations. Each amino acid In the CDR was mutated to histidine using the In-Fusion HD Cloning Kit (Clontech Inc. or Takara Bio company), respectively, according to the manufacturer's instructions. After confirming the correct mutation of each variant by sequencing, the variants were transiently expressed and purified by the following method: recombinant antibodies were transiently expressed using Freestyle FS293-F cells and 293Fectin (Life technologies) according to the manufacturer's instructions. The recombinant antibody was purified as protein A (GE healthcare) and eluted in D-PBS or His buffer (20mM histidine, 150mM NaCl, pH 6.0). Size exclusion chromatography is further performed to remove high molecular weight and/or low molecular weight components, if desired. All histidine-substituted variants are improved over the methods described aboveThe assay was evaluated. Briefly, immediately after the dissociation phase at pH7.4, another dissociation phase at pH5.8 was integrated And (6) analyzing. This was done to assess pH-dependent dissociation of the complex formed between antibody (Ab) and antigen (Ag) at pH7.4, rather than the corresponding dissociation at pH 5.8. The dissociation rate at pH5.8 buffer was determined by processing and fitting the data using Scrubber 2.0(BioLogic Software) curve fitting Software.

Selection and combination resulted in a pH of 5 as compared to the pH7.4 dissociation phaseA single histidine substitution with reduced binding at 8. To identify mutations that improve affinity at ph7.4, more than 500 variants were generated for the heavy and light chains, respectively, using at least one of the variants generated in the histidine substitution step. Each amino acid in the CDRs of these variants was substituted with 18 other amino acids, except for the original amino acid and cysteine. Use ofThe 4000 apparatus (GE Healthcare) assessed variants for their ability to bind to human CCL2 at 37 ℃ at pH 7.4. As previously described, immediately after the dissociation phase at pH7.4, another dissociation phase at pH5.8 is integratedAnd (4) measuring. The dissociation rate at pH5.8 buffer was determined by processing and fitting the data using Scrubber 2.0(BioLogic Software) curve fitting Software.

Variants with improved pH7.4 affinity and improved pH dependence were selected and these mutations were combined. In the table below, four 11K2 variants and four CNTO888 variants are exemplified.

Table (b): four modified 11K2 and four CNTO888 variants designed for pH-dependent binding

Modified 11K2 variants (VH/VL)	Modified CNTO888 variants (VH/VL)
		11K2H1503/11K2L1338	CNTO888H0625/CNTO888L0616
11K2H1510/11K2L1338	CNTO888H0634/CNTO888L0616
		11K2H1503/11K2L1201	CNTO888H0635/CNTO888L0616
11K2H1514/11K2L1338	CNTO888H0695/CNTO888L0616

To evaluate the combined effect of the modified 11K2 and CNTO888 variants, each 11K2 variant was combined with four modified CNTO888 variants and expressed as a biparatopic CCL2 antibody in the CrossMab format. Illustrated in the table below, where the 4 x 4 combination resulted in the production of 16 biparatopic antibodies, designated CKLO01 through CKLO 16.

Table: the combination of four modified 11K2 and four modified CNTO888 variants yielded 16 bispecific (biparatopic) antibodies.

To generate bispecific antibodies, the CrossMab technique described in WO 2016/016299 was used, in which VH/VL had been exchanged in one antibody arm, while the CH1/CL interface of the other antibody arm had been modified by charge modification, in combination with the knob-into-holes (knobs-into-holes) technique in the CH3/CH3 interface to promote heterodimerization. Exemplary sequences for all four antibody chains applying this technique are provided for CKLO2 IgG1 (see SEQ ID NO: 108 through SEQ ID NO: 111). Depending on the heavy chain constant domain used (e.g., IgG1 wild-type (no Fc receptor binding silent mutation), PGLALA, SG1095, SG1099, 1100-for SG1095, SG1099, 1100, see description below or sequence description), suffixes IgG1, PGLALA, SG1095, SG1099, 1100

Biparatopic anti-CCL 2 antibodies with modified variable domains and CDRs (ion-dependent/pH-dependent binding) Functional characterization of bodies

Affinity measurements (see methods above)

For all 16 generated bispecific anti-CCL 2 antibodies (all IgG1 wild-type), their pH-dependent binding to human CCL2 was determined.

FIG. 5a showsSensorgram showing binding curves of four modified 11K2 and four CNTO888 variants and, in combination, 16 crosssmabs to monomeric CCL2 at ph7.4 (black line) and ph5.8 (grey line).

FIG. 5b showsSensorgram showing the binding curves of four modified 11K2 and four CNTO888 variants and 16 crosssmabs after combination with monomeric CCL2, wherein another dissociation phase at ph5.8 was integrated into the dissociation phase at ph7.4 immediately after the dissociation phaseAnd (4) measuring.

Cross-reactive binding of CCL8

The pH dependence of binding to recombinant monomeric human CCL2 and recombinant monomeric human CCL8 was assessed at 37 ℃ using a Biacore T200 instrument (GE Healthcare). Anti-human fc (GE Healthcare) was immobilized on each flow cell of the CM4 sensor chip using an amine coupling kit (GE Healthcare) according to the manufacturer's recommended settings. The antibody and analyte were diluted into ACES pH7.4 or pH5.8 buffer (20mM ACES, 150mM NaCl, 1mg/ml BSA, 0.05% Tween 20, 0.005% NaN 3). Antibodies were captured on an anti-Fc sensor surface and recombinant monomeric human CCL2 was then injected onto the flow cell at a concentration of 8 nM. The association phase of the analyte with the antibody was monitored for 120 seconds, followed by a 180 second dissociation phase. Using 3M MgCl for each cycle ₂ The sensor surface is regenerated. The binding sensorgrams were treated with TIBCO spirovir by normalization of the binding response to the capture level.

The pH-dependent dissociation assessment of the antibody/antigen complexes formed at pH 7.4 was examined by a modified Biacore assay. Briefly, immediately after the dissociation phase at pH 7.4, another dissociation phase at pH 5.8 was integrated into the Biacore assay. The binding sensorgrams were treated with TIBCO spirovir by normalization of the binding response to the capture level.

Expression and purification of recombinant human CCL 8P 8A monomer: sequence of wild-type human CCL8 obtained from Genbank (NCBI: NP-005614.2) to prepare monomeric CCL8, proline at position 8 of the mature CCL8 protein was mutated to alanine. Expi 293 cells (Lifetech) were transfected according to the manufacturer's instructions. The CCL8 wild-type and P8A monomeric proteins were purified from cell culture supernatants by cation exchange chromatography using SP-Sepharose HP (GE Healthcare) and Superose 200 size exclusion (GE Healthcare) chromatography using the same method. Briefly, cell culture supernatants were diluted 2.5-fold with MilliQ water (Millipore), loaded onto a Hi-Trap SP-HP column equilibrated with PBS, washed with equilibration buffer, and eluted using a 0-2M NaCl gradient. The eluted protein fractions were combined and further purified by size exclusion chromatography, equilibrated with 20mM histidine, 150mM NaCl, pH 6.0, using a HiLoad 16/600Superose 200(GE Healthcare) column. Fractions were analyzed by size exclusion chromatography and SDS-PAGE. Fractions containing CCL8 protein were pooled, concentrated and stored at-80 deg.C

Human CCL8 has high homology with CCL2 and also binds CCR 2. Since the 11K2 arm was able to bind CCL8 (see fig. 1), it was necessary to identify mutations to reduce this binding, avoiding off-target effects of neutralizing CCL 8. Furthermore, removal of CCL8 binding on the 11K2 arm was important for efficient formation of immune complexes with CCL 2. Since the CNTO arm does not bind CCL8, the binding of CCL8 to the 11K2 arm will interfere with the immune complex formed with CCL2, which may reduce the clearance of CCL2 from plasma.

To identify mutations that reduce binding of 11K2 to human CCL8 and confer selectivity to human CCL2, some CDR positions were substituted, such as D101E in the 11K2 VH of CKLO02 or W92R in the 11K2 VL of CKLO03, to remove cross-reactivity with huCCL 8. As shown in fig. 6, CCL8 binding in the double paratope crosssmab can be significantly reduced by engineering 11K 2. The CKLO01 variants were not optimized to reduce CCL8 binding, whereas CKLO04, CKLO03 and CKLO02 comprise mutations to reduce CCL8 binding. All four crosssmabs had pH dependent binding to CCL 8.

Binding affinities of anti-CCL 2 antibodies to recombinant CCL2 and CCL8 at pH7.4 and pH5.8

To determine the affinity and pH-dependent binding of parental CNTO888H/11K2H2, binding of CKLO1, CKLO2 and CKLO3 to human CCL2 and CCL8 was assessed at 37 ℃ using a Biacore T200 instrument (GE Healthcare). Anti-human fc (GE Healthcare) was immobilized on each flow cell of the CM4 sensor chip using an amine coupling kit (GE Healthcare) according to the manufacturer's recommended settings. The antibody and analyte were diluted into ACES pH7.4 or pH5.8 buffer (20mM ACES, 150mM NaCl, 1mg/ml BSA, 0.05% Tween 20, 0.005% NaN 3). Antibodies were captured onto anti-Fc sensor surfaces and recombinant human CCL 2P 8A variant (monomer) or CCL 8P 8A variant (monomer) was injected into the flow cell at 1.25nM to 20nM prepared in two-fold serial dilutions. The sensor surface was regenerated with 3M MgCl2 every cycle. Binding affinity was determined by processing the data using Biacore T200 evaluation software version 2.0 (GE Healthcare) and fitting it to a 1:1 binding model. The binding affinities of anti-CCL 2 antibodies to recombinant CCL2 and CCL8 at pH7.4 and pH5.8 are shown in table 5 below.

Table 5: binding affinity of anti-CCL 2 antibody to recombinant CCL2 and CCL8 at pH7.4 and pH 5.8

Note that: n.d. KD cannot be determined due to low binding reactions.

The data in table 5 show that for CKLO2 and CKLO3, binding to CCL8 was abolished at pH7.4 while retaining strong affinity and pH-dependent binding to CCL2 at pH 7.4. The results show that affinity matured variants CKLO1, CKLO2, CKLO3 were successfully generated based on different modifications introduced in the variable regions and CDRs of the parent bispecific antibody of CNTO888 and 11K2, with enhanced binding affinity to CCL2 compared to the parent Ab at pH 7.4. Meanwhile, CKLO1, CKLO2, CKLO3 showed strong pH-dependent binding to CCL 2. The binding affinity of CCL2 was 1000-fold or more weaker in KD at pH 5.8 than at pH7.4

Clearance of wild type human CCL2

To assess the ability of pH-dependent bispecific antibodies to enhance clearance of wild-type human CCL2, preformed immune complexes consisting of anti-CCL 2 single paratope antibody (20mg/kg) and wild-type human CCL2(0.1mg/kg) were administered in a single dose of 10ml/kg into the tail vein of SCID mice. Blood was collected 5 minutes, 1 hour, 4 hours, 7 hours, 1 day, and 7 days after administration. The serum was stored at-80 ℃ or below-80 ℃ until measured. The Crossmab antibodies tested were the parent CNTO//11K2, and the four pH engineered variants CKLO01, CKLO02, CKLO03, and CKLO 04. All antibodies had IgG1 wild-type Fc portion (no mutation silencing/eliminating Fc (γ) receptor binding). Measurements of total human CCL2 and anti-CCL 2 antibody concentrations in mouse serum were performed as described above (after table 4, "evaluation of human CCL2 immune complexes cleared with biparatopic antibodies in mice").

The results are shown in FIG. 7 a: pre-formed immunocomplexes consisting of hCCL2 and bispecific anti-CCL 2 antibodies (parental CNTO//11K2 and pH-dependent variants CKLO01, CKLO02, CKLO03, and CKLO04) were injected to hCCL2 serum concentrations for a period of time after SCID mice. All four of these pH engineered variants showed rapid clearance of human CCL 2. For both CKLO02, CKLO03, human CCL2 was below the detection limit on day 1. For parent CNTO//11K2, rapid clearance of human CCL2 was initially observed until day 1, but after that, clearance of human CCL2 was slow.

Example B-3 anti-CCL 2 antibody with modified variable domains and CDRs (ion-dependent/pH-dependent binding) and Fc-mediated clearance

Modification of bispecific anti-CCL 2 antibodies by scavenging techniques

Bispecific anti-CCL 2 antibodies were modified using clearance techniques such that the bispecific anti-CCL 2 antibody was able to eliminate free CCL2 for longer periods of time, thereby being able to sustain biological effects in vivo, such as anti-cancer efficacy or anti-inflammatory efficacy.

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

pI Fc mediated clearance

We have demonstrated that pH engineered biparatopic antibodies can accelerate CCL2 clearance in vivo, and we next evaluated antibodies with pI-increasing substitutions for the ability to enhance clearance of wild-type human CCL 2. Preformed immune complexes consisting of anti-CCL 2 monoparate antibody (20mg/kg) and wild-type human CCL2(0.1mg/kg) were administered to the tail vein of SCID mice in a single dose of 10 ml/kg. Blood was collected 5 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 7 days after administration. The serum was stored at-80 ℃ or below-80 ℃ until measured. The Crossmab antibodies tested were parental CKLO03 with IgG1 and CKLO03, CKLO03-SG1099 with pI enhancing Fc. Measurements of total human CCL2 and anti-CCL 2 antibody concentrations in mouse serum were performed as described above (after table 4, "evaluation of human CCL2 immune complexes cleared with biparatopic antibodies in mice").

The results are shown in FIG. 7 b: pre-formed immunocomplexes consisting of hCCL2 and CKLO03 (with IgG1 wild-type Fc) or CKLO03-SG1099 (with enhanced I Fc CKLO03) were injected into hCCL2 serum concentrations during the latter period of SCID mice. CKLO03-SG1099 containing the Fc substitution Q311R/P343R (EU Kabat numbering) showed faster clearance/decrease in human CCL2 compared to CKLO03 with IgG 1. This suggests that a pH-dependent biparatopic antibody with a pI-increasing mutation may accelerate the clearance of CCL 2.

Generation of biparatopic anti-CCL 2 antibodies with Fc γ RIIb enhanced Fc variants and other Fc modifications

In this example, Fc engineering to enhance CCL2 clearance is illustrated.

It has been demonstrated in WO 2013/125667 that the clearance of soluble antigens can be enhanced by administering antigen binding molecules (e.g. antibodies) comprising an Fc domain exhibiting increased affinity for Fc γ RIIb. Furthermore, Fc variants have been described in WO 2012/115241 and WO 2014/030728 that can show enhanced binding of human Fc γ RIIb. It has also been demonstrated that these Fc variants can show selectively enhanced binding to human fcyriib and reduced binding to other active fcgamma receptors (fcyrs). This selective enhancement of fcgrriib binding not only facilitates clearance of soluble antigens, but also helps to reduce the risk of undesired effector function and immune response.

To develop antibody drugs, efficacy, pharmacokinetics, and safety should be evaluated in non-human animals with the drug having pharmaceutical activity. Other approaches, such as the use of surrogate antibodies (int.j. tox.28:230-253(2009)), must be considered if active only in humans. However, it is not easy to use alternative antibodies to accurately predict the effect of the interaction between the Fc region and Fc γ R in humans, because the expression pattern and/or function of Fc γ R in non-human animals is not always the same as in humans. The Fc region of preferred antibody drugs should be cross-reactive with non-human animals, particularly cynomolgus monkeys, which have an expression pattern and function close to that of human Fc γ Rs, so that the results obtained in non-human animals can be extrapolated to humans.

The following IgG1 constant domain/Fc variants of bispecific anti-CCL 2 antibody were generated with mutations at positions in the Fc portion (EU Kabat numbering)

SG 1095-derived from IgG1, including mutations (Kabat EU numbering):

L235W/G236N/H268D/Q295L/a330K/K326T (suitable for increasing affinity for human FcgRIIb and for decreasing affinity for other human FcgR);

Q438R/S440E (suitable for inhibiting rheumatoid factor binding)

SG1099 from IgG1 includes mutations (Kabat EU numbering):

Q311R/P343R (adapted to increase pI to enhance antigen uptake)

SG 1100-derived from IgG1, including mutations (Kabat EU numbering):

-Q311R/P343R (adapted to increase pI to enhance antigen uptake);

Q438R/S440E (suitable for inhibiting rheumatoid factor binding)

GG01, including mutations (Kabat EU numbering) derived from IgG 1:

Q438R/S440E (suitable for inhibiting rheumatoid factor binding)

GG02, including mutations (Kabat EU numbering) derived from IgG 1:

Q438R/S440E (suitable for inhibiting rheumatoid factor binding)

GG 03-derived from IgG1(SG1-IgG1 allotype), including mutations (Kabat EU numbering):

Q438R/S440E (suitable for inhibiting rheumatoid factor binding)

GG 04-derived from IgG1(SG1-IgG1 allotype), including mutations (Kabat EU numbering):

-Q438R/S440E (suitable for inhibiting rheumatoid factor binding)

With modified variable domains and CDRs (ion-dependent/pH-dependent binding) and with or without Fc mediation Functional characterization of the cleared bispecific anti-CCL 2 antibody of (a)

SPR binding of Fc variants SG1095, GG01, GG02, GG03/04 of CKLO2

Binding of monomeric human CCL2 and cynomolgus monkey CCL2 to 4 different antibodies P1AD8325, P1AF8137, P1AF8139 and P1AF8140 at pH 7.4 and 5.8 was studied in SPR assays performed on a Biacore 8K instrument.

In this setup, CaptureSelect was coupled using an amine coupling method ^TM Human Fab- κ (ThermoFisher Scientific) was immobilized on a CM3 sensor chip, capturing various antibodies as ligands, and performing measurements at two different pH values with 0, 10, 100 and 1000nM monomeric Human CCL2 or cynomolgus CCL2 as analytes.

CKLO2-SG1095, CKLO2-GG01, CKLO2-GG02, CKLO2-GG03/04 show almost the same binding curves as monomeric human CCL2 and cynomolgus monkey CCL2, which bind at 10, 100 and 1000nM and dissociate equally rapidly at pH 7.4, whereas no stable binding is observed at pH 5.8. The results are shown in the table below.

Table: the combination of the monomer human CCL2 and the cynomolgus monkey CCL2 with CKLO2-SG1095, CKLO2-GG01, CKLO2-GG02 and CKLO2-GG03/04 shows almost the same combination curve as the monomer human CCL2 and the cynomolgus monkey CCL2

Chemotaxis assay

Description of the method

THP-1 cells were cultured for 3 days until 8X10E5 cells/ml. A total cell number of 5000 cells/well was seeded in the upper chamber of the microtiter dish and allowed to settle at 37 ℃, and recombinant huCCL2 was pipetted into the bottom chamber at a final concentration of 50ng/ml (100 ng/ml for recombinant mucccl 2 when assays were performed to test surrogate antibodies) with or without anti-CCL 2 antibody. The top and bottom chambers were pooled together to avoid air bubbles, the plates were then incubated at 37 ℃ for 24 hours, the migrated cells were quantified by the Cell Titer Glo method according to the manufacturer's recommendations, and luminescence was measured using a Tecan Infinite 200 Reader.

The results are shown in FIG. 8: chemotaxis assay: bispecific anti-CCL 2 antibodies with identical CDRs and variable regions VH/VL, i.e., CKLO2-IgG1 wild-type and CKLO2-SG1095, but with different Fc portions, can be of identical potency (IC) ₅₀ 0.2 μ g/ml; fig. 8, left panel) inhibits THP-1 cell migration.

Similarly, CCL2-0048 (parent VH/VL-unmodified bispecific antibody CNTO888/11k2 of CKLO2, which is pH independent) also showed IC ₅₀ 0.2. mu.g/ml, since pH dependence is crucial for antigen clearance, a phenomenon that does not occur in this assay.

The corresponding single paratope antibody CNTO888 IgG1 and humanized 11k2 IgG1 showed IC ₅₀ Values were 0.3 and 0.7. mu.g/ml, respectively, whereas the huIgG1 isotype control showed no inhibition (FIG. 8, right panel).

In other similar experiments, IC's of CKLO2-GG01 (0.2. mu.g/ml), CKLO2-GG02 (0.2. mu.g/ml) and GG03/GG04 (0.3. mu.g/ml) were determined ₅₀ The value is obtained.

In vivo biological Activity in transgenic mouse model

Materials and methods

B16-huCCL2/CCL 2-hypothetical model

The purpose of generating the cost model was to test anti-human CCL2 antibodies without interfering with mouse CCL2 in immunocompetent tumor-bearing mice. For this reason, the mouse tumor cell line B16-F10 was selected because it did not secrete mCCL2 and is known to grow in mice of the C57/Bl6 line, which is the genetic background of CCL2 knockout mice.

To generate a stable pool of huCCL 2-expressing B16F10 tumor cells, they were transfected with plasmid DNA encoding huCCL2 and a hygromycin B (hygromycin B) selection cassette. Thus, cells were seeded at a density of 2.0E +05 cells/well in growth medium (DMEM + 10% FCS +2mM L-glutamine) in 6-well plates. After 24 hours, a transfection mixture consisting of 1. mu.g DNA per well and Opti-MEM medium containing Lipofectamine 2000 was added. Subsequently, the cells were selected with a medium containing hygromycin-B (0.5 mg/ml). After 20 days of culture, viable single cells were sorted using BD FACS Aria III based on FSC/SSC-scattering. After 12 days, cell culture supernatants from single cell clones were screened for expression of human CCL2 using ELISA Ready-SET-Go from ebioscience (Cat #88-7399-86) compared to wild-type B16F10 cells (data not shown).

Selected B16-F10_ HOMSA _ CCL2 tumor cells selected clones 1a5, 2A3, and 2B2 were routinely cultured in DMEM containing 10% FCS and 2mM L-glutamine (PAN Biotech GmbH, Germany) at 37% water saturation pressure at 5% CO 2. The culture subcultures were performed twice weekly in trypsin/EDTA 1x (PAN Biotech GmbH, Germany).

Female B6.129S4-Ccl2tm1Rol/J mice (Jackson Laboratories) reached 7 to 10 weeks of age, inoculated with a B16-F10_ HOMSA _ CCL2 tumor cell clone: on the day (study day 0), tumor cells were harvested from culture flasks, transferred to medium, washed once and resuspended in PBS. The CELL number was determined using a CELL counter and analyzer system (Vi-CELL, Beckman Coulter). The cell titer for s.c. injection was adjusted to 1 × 10E7 cells/ml and injected subcutaneously into the right flank of mice at 100 μ l using a cooled 1.0ml tuberculin syringe (Dispomed, Germany) and a small needle (0.45 × 12 mm). Cell seeding was performed under general anesthesia with isoflurane (isoflurane) (CP Pharma, germany) in inhalation units of small animals.

Tumor growth was monitored daily and mice were sacrificed on study day 15 when B16-F10_ HOMSA _ CCL2 tumor cells clones 1a5 and 2A3 reached about 1000mm3 (at this time, the 2B2 tumor was about 600mm3 due to the slower growth rate). At endpoint, blood samples were collected for CCL2 measurement as described above, and tumors were removed and analyzed by flow cytometry.

The finding that mouse immune cells infiltrated all tumors confirmed the notion that human CCL2 was able to attract mouse CCR2+ cells. B16-F10_ HOMSA _ CCL2 tumor cells clone 1a5 showed the highest total infiltration rate of CD45+ and the highest relative mdscs (monocyte-derived suppressor cells) composition (fig. 2). Even though 2A3 cells caused serum total CCL2 levels similar to 1a5 cells, clones 2A3 and 2B2 had lower immune cell frequencies in tumors, while 2B2 clone showed significantly lower CCL2 serum concentrations (data not shown).

Female B6.129S4-Ccl2tm1Rol/J mice were inoculated with B16-F10_ HOMSA _ Ccl2 tumor cell clone 1a5, as described above.

Treatment of the study group was started 5 days after cell inoculation. Group 1 received human IgG vehicle control treatment, while groups 2 and 3 received Mab CKLO2-IgG1(Fc wild-type IgG1) and CKLO2-SG1099 ((CKLO 2 pI-enhanced Fc based on IgG1 with mutation Q311R/P343R (Kabat EU numbering)), at 3,7mg/kg daily for 9 days i.p. treatment, at day 14 of the study mice were sacrificed and tumors were transplanted, single cell suspensions were generated from each tumor mass using enzyme digestion and cell filters, non-pooling analysis was performed by flow cytometry, for detection of the immune cell population of interest, using the following markers and fluorescent dyes CD45-BUV395, CD11b-BUV737, F4/80-421, CD11c-BV605, Ly6C-AF488, Ly 6G-CP-Cy5.5, CD 206-BV-686, CD 8-APC 686 8-APC 42-H11-H7-H11-H-34-H7-H-27-NO. 7375.3, CD279-APC and CD 274-PE. Samples were collected using a BD LSR-Fortessa flow cytometer and analyzed using BD Diva Software.

Serum samples were drawn at days 6, 8, 11, and 14 of the study to measure total huCCL2 and free huCCL 2.

The method for detecting free CCL2 is detailed below in "conceptual validation study of CCL2 clearance efficiency in cynomolgus macaques". To analyze free human CCL2 in this study, recombinant cynomolgus CCL2 was replaced with recombinant human CCL2 to prepare calibrators and QC.

Serum samples of total CCL2 were analyzed by an unverified but qualified specific sandwich ELISA. Briefly, biotinylated anti-CCL 2 capture antibody (CNTO888 CCL2-0004), blocking buffer, pre-treated test sample, and detection reagent (digoxiylated anti-CCL 2 antibody (M-1H11-IgG)) were added stepwise to 384-well streptavidin coated microtiter plates, and the plates were incubated in a non-vigorous shaker for 1 hour in each step. To dissociate CCL 2-drug complexes in the pretreatment step, calibrators or QCs were acidified at pH 5.5 for 10 min at 37 ℃. The acidified sample was added to the SA-MTP. To detect the immobilized immune complexes, polyclonal anti-digoxigenin-POD conjugates were added and the discs were incubated for 60 min. After each step, the dish was washed three times to remove unbound material.

ABTS was added to the plate and incubated with shaking at room temperature. The absorption was measured at a wavelength of 405/490 nm. Human CCL2 concentration was calculated based on the response of the calibration curve using analytical software xlfit (idbs).

Depending on the statistically analyzed data set, a t-test or one-way ANOVA and Tukey test were applied accordingly for multiple comparisons.

As a result, the

At the end of the study, tumor volume and tumor weight were significantly reduced in those mice that received CKLO2-SG1099(CKLO2 pI-enhanced Fc) (fig. 9). Tumor infiltration was carefully observed and found to be reduced by mononuclear myeloid-derived suppressor cells (M-MDSCs), as expected by CCL2 blockade (fig. 9).

In addition, serum analysis confirmed the effectiveness of the therapy: pI optimization did result in a reduction in total CCL2 accumulation compared to IgG1 wild-type Fc CKLO2 molecule, whereas free CCL2 (not bound to antibody) was completely inhibited at the detection limit (fig. 10). Thus, this model is suitable for studying the effect of CCL2 blockade in tumors using the anti-huCCL 2 biparatopic clearing antibody CKLO2-SG1099(CKLO2 pI-enhanced Fc). The optimal dose regimen was studied in subsequent studies by administering CKLO2-SG1099(CKLO2 pI-enhanced Fc) at lower doses once or twice weekly and monitoring the spread of inhibition by free CCL2 over several weeks. In addition, combinations with T cell activation therapy (i.e. T cell bispecific, PD-L1 blocking) were also explored in this model.

In additional similar experiments, other variants were analyzed, such as CKLO2-SG1095, CKLO2-GG01, GG02 and GG03/GG 04.

Concept validation (POC) study of CCL2 clearance efficiency of cynomolgus macaques

A method. The primary objective of this study was to evaluate the expansion of CCL2 inhibition in cynomolgus macaques and the efficiency of clearance of four anti-CCL 2(MCP-1) antibodies. A secondary objective was to evaluate the Pharmacokinetic (PK) profile of these antibodies. All antibodies were administered to all 3 to 4 year old males within 30 minutes as a single IV infusion of 25mg/kg and total CCL2 and antibody concentrations in serum were measured over 70 days. The anti-CCL 2 antibodies studied consisted of control antibodies (group 1 and group 2) and antibodies specifically engineered to enhance elimination of the CCL 2-drug complex (hereinafter referred to as antigen clearance or simply clearance). Group 1: CNTO 888-SG 1(═ IgG1 wild-type) anti-CCL 2 antibody (n ═ 3 animals) as a control for maximum total accumulation of CCL 2; group 2: a biparatopic anti-CCL 2 antibody CKLO2-SG1(IgG1 wild-type) (n-3) with pH-dependent target binding but no Fc modification; group 3: biparatopic anti-CCL 2 antibody CKLO2-SG1100 (n-4) with pH-dependent target binding and Fc-pI and further modified and group 4: a biparatopic anti-CCL 2 antibody CKLO2-SG1095 (n-4) with pH-dependent target binding, Fc-pI Fc γ RII and further modifications.

In this study, total serum concentration of antibodies, total (free and antibody-bound CCL2), and free target were quantified. In addition, the presence of anti-drug antibodies (ADA) was assessed. Antibody, total CCL2 and free CCL2 profiles were analyzed by non-compartmental analysis using Phoenix 64 (Pharsight/Certara); data are illustrated using GraphPad Prism v.6.07(GraphPad Software).

For antibodies, serum samples were analyzed using a general human sandwich ELISA method. The concentration of total antibodies in monkey sera was measured by ELISA. mu.g/mL anti-human kappa chain antibody was immobilized on maxisorp 96-well plates overnight and then incubated in blocking buffer at 30 ℃ for 2 hours. Antibody calibration curve samples, quality control samples and monkey serum samples were incubated on the dish for 1 hour at 30 ℃ prior to washing. Next, anti-human IgG-HRP was added and incubated at 30 ℃ for 1 hour before washing. ABTS substrate was incubated for 10, 20 and 30 minutes and then detected with a microplate reader at 405 nm. Antibody concentrations were calculated based on the response of the calibration curve using the analytical software SOFTmax PRO (Molecular Devices).

A non-validated but qualified specific sandwich ECL method assay was used to analyze total CCL2 serum samples. anti-CCL 2 antibody (r2F2-SG1) at 3. mu.g/mL was immobilized on a Multi-ARRAY 96-well plate (Meso Scale Discovery) overnight and then incubated in blocking buffer at 30 ℃ for 2 hours. The cynomolgus CCL2 calibration curve sample, quality control sample, and cynomolgus diluted serum sample were incubated with ph5.5 acidic buffer at 37 ℃ for 10 minutes. Thereafter, the samples were incubated on anti-CCL 2 fixed disks at 30 ℃ for 1 hour, and then washed. Next, the SULFO TAG-labeled MCP-1 antibody was added and incubated at 30 ℃ for 1 hour before washing. Read Buffer T (x4) (Meso Scale Discovery) was immediately added to the disc and the signal was detected with SECTOR Imager 2400(Meso Scale Discovery). The concentration of cynomolgus CCL2 was calculated based on the response of the calibration curve using the analytical software SOFTmax PRO (Molecular Devices).

Serum samples of free CCL2 were analyzed in an unverified but qualified Gyrolab (tm) immunoassay run on Gyrolab xpore. Biotinylated anti-CCL 2 antibody (M-2F6-IgG) was used as capture reagent and Alexa647 labeled anti-CCL 2 antibody (M-1H11-IgG) was selected for detection. Both reagents were diluted to 1 μ g/mL with PBS, 0.1% Tween, 1% BSA and then transferred to 96-well PCR disks (Fisher Scientific). Cynomolgus CCL2 calibration curve samples, QC and undiluted serum samples were also transferred to 96-well PCR plates. Both disks were loaded into the instrument together with a Gyrolab Bioafy 200nl disk (Gyross Protein Technologies AB). A three step assay protocol (200-3W-001) was chosen. Briefly, the protocol describes the sequential addition of capture, sample and detection reagents to a designated streptavidin column of Gyrolab Bio Affy 200 discs, each reagent arriving simultaneously on the column after a short spinning step on the disc, washing the column with 0.05% Tween PBS after each step, and finally recording the laser-induced fluorescence values in the instrument. The concentration of free cynomolgus CCL2 was calculated based on the response of the calibration curve using XL Fit software (IDBS).

ADA was analyzed using the method described elsewhere (Stubenrauch et al, 2010). In summary, biotinylated mAb anti-human Fc γ -pan R10Z8E9 was bound to streptavidin-coated high binding plates at a concentration of 0.5 μ g/mL and incubated for 1 hour, samples and standards were diluted with assay buffer to 5% cynomolgus monkey serum and added to each well of the coated plates after washing and incubated for 1 hour with shaking. After washing, 0.1. mu.g/mL digoxin anti-cynomolgus monkey (cyno) IgG was added and incubated for 1 hour with shaking. After washing, 25mU/mL of polyclonal anti-digoxigenin-HRP conjugate was added and the incubation was shaken for 1 hour. ABTS was added to the disc and incubated with shaking for 10 minutes at room temperature. To measure the absorption with a microplate reader at a wavelength of 405/490 nm. ADA concentrations were calculated based on the response of the calibration curve using analytical software SOFTmax PRO (Molecular Devices).

And (4) obtaining the result. PK behavior was assessed over time in animals without ADA (i.e., before day 14). The serum concentration-time curves for all anti-CCL 2 antibodies were similar during this period (see fig. 11, left panel), and the partial mean AUC values (AUC) were found between the different groups of 1490, 1810, 1210, and 1320 days μ g/mL in groups 1, 2, 3, and 4, respectively _0-7d ) And (4) the equivalent. Similarly, average C _max Values are comparable to 620, 764, 616 and 664. mu.g/mL for groups 1, 2, 4 and 4, respectively. The extent of ADA development varied widely from animal to group and resulted in highly variable PK profiles after day 7 (data not shown). One animal in group 2 was ADA negative throughout the 70 day observation period (see figure 11, right panel). In this animal, the clearance, distribution volume and terminal half-life of the anti-CCL 2 antibody were 7.34 mL/(day. kg) by non-compartmental analysis,76.2mL/kg and 10.9 days.

Baseline levels of CCL2 in serum were assessed in each animal before antibody treatment began. Basal CCL2 levels ranged from 0.126 to 0.357ng/mL (geometric mean (% CV): 0.199ng/mL (32.2%, N ═ 14)). Since the free form of CCL2 had a higher elimination rate than the antibody-bound form of CCL2, an increase in total CCL2 serum concentration was expected after antibody treatment. This was indeed observed in all groups (left panel of fig. 12), indicating that all antibodies bound to the target. C of Total CCL2 in groups 1, 2, 3, and 4 under treatment _max Values were increased to 824, 575, 106, 32.7ng/mL, respectively. AUC of groups 1, 2, 3 and 4 _0-7d Values were 3060, 2970, 522 and 181 days ng/mL, respectively. During the time the animals were ADA negative, the molar drug concentration still exceeded the total CCL2 concentration. Two clearing anti-CCL 2 antibodies (groups 3 and 4) showed serum C-based serum C at total CCL2 serum concentrations compared to the conventional antibody (group 1) _max Values were about 8-fold and 25-fold, respectively, and AUC based on total CCL2 _0-7d A considerable reduction of the value by a factor of about 6 and 17. ADA negative animals in group 2 showed sustained target engagement (plateau regions evident) of total CCL2 concentration (fig. 12 right panel).

Treatment with all antibodies resulted in a significant reduction in serum levels of free CCL2 (left panel in fig. 13), but this reduction was only in part at the beginning. In group 1, all individuals again reached quantifiable levels of free CCL2 after one day. In group 2, all individuals again reached quantifiable levels of free CCL2 after two days. In groups 3 and 4, two individuals per group showed seven days of inhibition of free CCL 2. In group 3, two animals with moderate ADA response and maintained adequate antibody concentration showed free CCL2 inhibition within the 21 day detection limit. In ADA positive animals, antibody depletion increased significantly and CCL2 levels quickly returned to their original baseline as a result of the loss of target binding (not shown here).

In additional similar experiments, other variants were analyzed, such as CKLO2-SG1099, CKLO2-GG01, GG02, and GG03/GG 04.

PK/PD research on CCL2 clearing efficiency of cynomolgus macaques

Study outline and goal. The PK/PD study was designed based on the results of POC studies performed using anti-CCL 2 antibody CKL02-SG 1095. Since the POC study demonstrated high anti-drug antibody (ADA) formation, it was added to the PK/PD study(obinitizumab) therapy, with the aim of reducing ADA response. For this purpose, Gazyva was administered four times by intravenous infusion at a dose of 30mg/kg throughout the study: days-14, -7, 8 and 36. On day 1 (groups 1-3), four animals per dose group (2/2 males and females) were dosed with CKL02-SG1095 at 2.5, 10 and 25mg/kg by IV infusion over 30 minutes. For comparison, 25mg/kg of conventional anti-CCL 2 antibody (CNTO888-IgG1) (group 4; same control as group 1 of the POC study described above) was IV infused over 30 minutes on day 1. Concentrations of total PK (CKL02-SG1095), total CCL2, and free CCL2 were assessed up to day 99 (i.e., 14 weeks post-dose).

The objective of the PK/PD study was to demonstrate an extended period of inhibition of free CCL2 by CKL02-SG1095 compared to conventional anti-CCL 2 antibody (CNTO888 with wild-type IgG1 Fc portion) in non-human primates.

A method. In this study, total PK and total CCL2 and free CCL2 were quantified. However, due to the presence of Gazyva in the serum samples, some corrections were made to the total PK assay, total CCL2 assay, and ADA assay compared to the POC study described herein. For CNTO888 IgG1, a PK analysis was not developed.

The concentration of total antibody CKL02-SG1095 in monkey sera was measured by ELISA. For ELISA biotinylated recombinant human CCL2 (antigen), pre-treated test samples, positive control standards (calibrator) or QC (quality control) and digoxin anti-human IgG (M-1.19.31-IgG) were added sequentially to 384-well streptavidin coated microtiter plates (SA-MTP). To dissociate the CCL 2-drug complex, a pre-treatment of the test sample was performed at pH 5.5 for 20 minutes. The acidified sample was neutralized prior to addition of SA-MTP. The immobilized immune complexes were detected with a polyclonal anti-digoxin-POD conjugate. After each step, the dish was washed three times to remove unbound material. ABTS was added as substrate to the disc and incubated at room temperature. The absorption was measured at a wavelength of 405/490 nm. Antibody concentrations were calculated based on the response of the calibration curve using analytical software xlfit (idbs).

Serum samples of total CCL2 were analyzed by an unverified but qualified specific sandwich ELISA. Briefly, biotinylated anti-CCL 2 capture antibody, pre-treated test sample, and detection reagent (digoxin anti-CCL 2 antibody (1H11-IgG1)) were added stepwise to 384-well anti-streptavidin coated microtiter plates and incubated on a shaker without vigorous shaking for 1 hour of capture and sampling steps, respectively, and 50 minutes of detection reagent. To dissociate CCL 2-drug complexes in the pretreatment step, calibrators or QCs were acidified at pH 5.5 for 20 min. The acidified sample was added to the SA-MTP. To detect the immobilized immune complexes, polyclonal anti-digoxigenin-POD conjugates were added and the discs were incubated for 50 minutes. After each step, the dish was washed three times to remove unbound material.

ABTS was added to the plate and incubated with shaking at room temperature. The absorption was measured at a wavelength of 405/490 nm. The cynomolgus CCL2 concentration was calculated based on the response of the calibration curve using analytical software xlfit (idbs). Capture antibodies for analysis of groups 1 to 3: anti-CCL 2 CNTO8888IgG1 for analysis of group 4 capture antibodies: anti-CCL 22F 2 IgG 1.

ADA was screened in 384-well plates using a bridging sandwich ELISA. Test samples, quality control samples and positive controls from group 1, group 2 and group 3 animals were incubated overnight at 500rpm on an MTP shaker at room temperature with biotinylated capture antibody CKL02-SG1095 and digoxigenin detection antibody CKL02-SG1095 and two other anti-CCL 2 antibodies (2F6-IgG1 and 1H11-IgG 1); these antibodies were added to neutralize CCL 2. For the group 4 animal samples, the biotin marker CNTO888-SG1 and digoxigenin CNTO888-SG1 were used, respectively. The formed immune complex was transferred to Streptavidin (SA) -coated MTP, and immobilized by biotin-labeled (Bi) capture antibody. After aspiration of the supernatant, unbound material was removed by repeated washing. Detection was accomplished by addition of anti-digoxin pod (p) conjugated antibody and ABTS substrate solution. The intensity of the color of the reaction (absorption at the reference wavelength at 405nm-490 nm) is determined by the brightness. If the signal is found to be above the specific cut point of the disc, the sample is defined as ADA positive. The cut points are defined during the analytical qualification.

And (6) obtaining the result. Despite the fact that(obinitizumab) pretreatment, but ADA developed in 10 and one-fourth of 12 animals treated with CKL02-SG1095 treated with CNTO888, had an effect on drug and biomarker concentrations. However, the two ADA negative animals of CKL02-SG1095 were in the 25mg/kg dose group and were directly comparable to the ADA negative animals of the CNTO888 group. PK behavior was assessed over time in animals without ADA (i.e., before day 10). PK profiles for three different dose groups of CKL02-SG1095 are shown in the left panel of fig. 14. Partial mean AUC values (AUC) for 2.5, 10 and 25mg/kg dose levels _0-7d ) 229/191 (male/female), 696/813 and 1492/1346 days. mu.g/mL, respectively. C for 2.5, 10 and 25mg/kg dose levels _max Values were 115/122 (male/female), 369/491 and 869/941. mu.g/mL, respectively. At the highest dose level, these findings were consistent with the POC study. For two ADA negative animals, the clearance, distribution volume and terminal half-life of CKL02-SG1095 were estimated by non-compartmental analysis to be 10.5-17.4 mL/(day. kg), 116-118mL/kg and 5.8-11.6 days, respectively (see FIG. 14, right panel).

For the POC study described above, accumulation of total CCL2 was observed after treatment with anti-CCL 2 antibody (see left panel of fig. 15). Baseline levels of CCL2 were assessed at five time points prior to dosing (included at (obinitizumab) one opportunity point before treatment); the average CCL2 baseline value is 0.742ng/mL, and(obinitizumab) treatment did not affect basal CCL2 levels. The extent of total CCL2 accumulation (values in parentheses indicate median fold change from baseline) depends on dose and constructionAnd (3) a body. For CKL02-SG1095, total CCL2 levels increased to 22.4(22), 67.2(105), and 54.9(76) ng/mL (median of four animals) at 2.5, 10, and 25mg/kg dose levels. For CNTO888 IgG1, the total CCL2 level increased to 1490(3160) ng/mL (median of 4 animals). Comparing ADA negative animals in groups 3 and 4, the cumulative level of CKL02-SG1095 was considerably lower compared to CNTO888 (fig. 15 right panel).

Treatment of all study groups resulted in a large transient decrease in serum free CCL2 levels (see left panel of figure 16; typically below the limit of detection (0.01 ng/mL)). For all ADA positive animals, free CCL2 levels quickly returned to baseline values after ADA formation (consistent with a reduction in drug exposure and rapid target transfer). For ADA negative animals (2/4 in group 3) and (3/4 in group 4), the free CCL2 inhibition period can be assessed throughout the study period. For the conventional antibody CNTO888 (panel 4), CCL2 inhibition period was short, presumably due to large accumulation of the total target (figure 15). Although drug concentrations were not quantified (no specific assay was available for CNTO 888), POC studies showed similar PK profiles between CNTO888 and CKL02-SG 1095. On the other hand, persistent inhibition of free CCL2 was observed for two ADA negative animals in group 3. For one animal, free CCL2 levels remained below the limit of detection within 29 days (right panel of fig. 16).

Prevalence studies of CCL2 in different tumor types

The following IHC prevalence studies of CCL2 and its receptor CCR2, including macrophage assays using CD163/CD68 and CD14, were performed on serum samples of 121 human matched tumors and 6 different indications:

pancreatic cancer (PaC)

Colorectal cancer (CRC)

Breast Cancer (BC)

Prostate cancer (PrC)

Ovarian cancer (OvC)

Stomach cancer (GC)

The following problems are solved:

do these tumors (hyper) express CCL 2?

Is CCL2 level elevated in the blood of these tumor patients?

Is there a correlation between blood and tumor CCL2 levels?

Tumor CCL2 and infiltrative CCR2 ⁺ Is there a correlation between immune cells?

Is there a correlation between tumor CCL2 and infiltrating myeloid cells?

Materials and methods

Histopathological scores were semi-quantitative. In addition, automated multiplex image analysis was used for CD163/CD68 IHC and tested for immune cell quantification of CD14 and CCR2 and CCL2 quantification.

Immunohistology studies were performed on resected specimens of 121 human tumors with 6 different indications: 31 pancreatic cancers (PaC), 30 colorectal cancers (CRC), 30 Breast Cancers (BC), 29 prostate cancers (PrC), 20 ovarian cancers (OvC) and 10 stomach cancers (GC) were provided by Indivumed (Hamburg) and Asterand (Royston/Herts, UK). Tumors were fixed in 4% formaldehyde buffer, paraffin embedded, cut to a thickness of 2.5 μm, and mounted on Superfrost Plus slides. Mouse monoclonal antibodies against CCL2 (clone 2D8, Novusbio NBP2-22115) were used on Ventana BXT following standard staining protocols (CC1 for 32', concentration 1. mu.g/mL in VBX, Optiview DAB detection System). Rabbit monoclonal antibodies against CCR2 (E68, Abcam ab32144) were used on Ventana Discovery XT following standard staining protocols (CC1 for 32', concentration 0.8ug/ml in DS2, Omni-UltraMap HRP DAB detection system). Mouse monoclonal antibodies against the monocyte marker CD14 (Cell Marque EPR3653, RTU) were used on the Ventana Discovery Ultra following standard staining protocols (CC1 for 64', Omni-UltraMap HRP DAB detection System). Macrophages and M2-like TAM (tumor-associated macrophages) CD163/CD68(DAB CD163 Mouse MRQ-26Cell Marqu) were applied on Ventana BXT according to standard staining protocols (CC1 for 32', CD163 RTU// CD68 concentration 0.6. mu.g/ml in DS2, detected with DAB and Red detection System) e RTU// red CD68 Mouse PG-M1 Dako). Ventana was used for all imagesA scan is performed. The tissue sections were subjected to semi-quantitative analysis.

1. Results

Prevalence of CCL2 and CCR2

All tumor indications analyzed showed that some tumors had variable levels of CCL2 upregulation and the presence of CCR2 on TAMs of variable amounts. (Table 6 below). For both CCL2 and CCR2, the highest expression was observed in ovarian cancer, followed by PDAC and GC

Characteristic features of tumour type

Tumors with high activity of CCL2-CCR2 were associated with high MDSC attraction and M2 polarized tumor growth enhanced immune status, representing preferred tumors for CCL2 blockade therapy. CCR2 IHC showed good correlation with MDSC and M2-like macrophages, confirming its biological role and confirming that this pathway has higher correlation as a biomarker compared to CCL2 IHC measurements. From the conclusions of this study, the following recommendations for CCL2 treatment can be concluded:

OvC is suggested for CCL2 targeted therapy due to the highest prevalence of CCL2 and CCR2 and the extent of M2 polarization;

because MDSCs are the highest in content compared to other analyzed tumor types, and because CCR2 and M2 are present at considerably higher levels and levels, PDACs may be recommended for CCL2 targeted therapy. CCL2 is also high in PDAC and is present much more in immune cells than in tumor cells than in other tumor types. PaC showed a good correlation between CCL2/CCR2 and MDSC attraction/M2 polarization. These results support the role of CCL2-CCR2 in analyzing PDACs, being highly focused on the attraction of immune cells.

For CCL2 targeted therapy, the use of Bc, in particular TNBC: BC showed considerably higher levels and contents of CCL2, CCR2, MDSC, and M2. Especially TNBC cases, characterized by higher M2 and MDSC content than non-TNBC, although non-TNBC showed the highest CCL2 production in tumor cells compared to other tumor indications.

The following tumor indications appear to be less dependent on the CCL2-CCR 2-axis and therefore may not be suggested for CCL2 targeted therapy:

CRC: CCL2 was low compared to other tumor types, especially compared to PaC. Also here, the lowest amount of M2-like macrophages was measured compared to other tumor types. The content of CCR2 and MDSC is variable. Interestingly, only in this indication a trend of positive correlation between CCL2 and CCR2 could be detected. However, overall findings support that the role of CCL2-CCR2 in CRC focuses on the survival of tumor cells rather than the attraction of immune cells.

Despite the higher CCL2, lower GC of CCR2 was observed, and the content of MDSCs was lowest. M2-like macrophages are present in varying amounts.

Table 6:in this study, CCL2 and CCR2 positive expression was analyzed in Tumor Cells (TC) and Immune Cells (IC) of different tumor types.

In PrC, CCR2 is present at variable levels. (unmeasured M2 and MDSC)

Correlation analysis

The results of the study can be summarized as follows:

between tumors CCL2 and CCR2(IHC), a positive correlation was present only in CRC.

Serum CCL2(ELISA) was not correlated with any of the measured parameters in tumors, including CCL2, CCR2, macrophages, and MDSCs. A trend positively correlated with tumor CCL2 was found only in PrC, both methods showed very low values.

CCR2 expression and M2-like macrophages and CD14 ⁺ The presence of cells was positively correlated with the ratio of M1/M2 and confirmed the biological role of CCR 2. The levels of CCR2 correlated better with M2 polarization compared to MDSC attractiveness.

CCL2 showed a trend of positive correlation with MDSC attraction and M2 polarization. Thus, CCL2 levels alone do not appear to be a major factor in the presence of MDSC and M2-like polarization.

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50 55 60

Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Glu

65 70 75 80

Ser Ala Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Gly Ala Thr Ile Ser

85 90 95

Tyr Leu Asp Asn Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys

100 105 110

<210> 17

<211> 6

<212> PRT

<213> Artificial

<220>

<223> heavy chain CDR-H11G 9

<400> 17

Leu Tyr Ser Tyr Met Cys

1 5

<210> 18

<211> 18

<212> PRT

<213> Artificial

<220>

<223> heavy chain CDR-H21G 9

<400> 18

Cys Val Asp Ala Gly Ala Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala

1 5 10 15

Lys Gly

<210> 19

<211> 19

<212> PRT

<213> Artificial

<220>

<223> heavy chain CDR-H31G 9

<400> 19

Gly Ile Leu Tyr Tyr Thr Trp Pro Tyr Pro Ala Gly Ala Ile Asp Ala

1 5 10 15

Phe Asp Ser

<210> 20

<211> 11

<212> PRT

<213> Artificial

<220>

<223> light chain CDR-L11G 9

<400> 20

Gln Ala Ser Glu Ser Ile Ser Asn Tyr Leu Ser

1 5 10

<210> 21

<211> 7

<212> PRT

<213> Artificial

<220>

<223> light chain CDR-L21G 9

<400> 21

Lys Ala Ser Thr Leu Ala Ser

1 5

<210> 22

<211> 12

<212> PRT

<213> Artificial

<220>

<223> light chain CDR-L31G 9

<400> 22

Gln Gln Ser Tyr Ser Ser Ser Asn Val Phe Asn Thr

1 5 10

<210> 23

<211> 128

<212> PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH 1G9

<400> 23

Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Ala Ser

1 5 10 15

Leu Thr Leu Thr Cys Lys Ala Ser Gly Ile Asp Phe Ser Leu Tyr Ser

20 25 30

Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Ala Cys Val Asp Ala Gly Ala Ser Gly Ser Thr Tyr Tyr Ala Ser Trp

50 55 60

Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Thr

65 70 75 80

Leu Gln Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Gly Ile Leu Tyr Tyr Thr Trp Pro Tyr Pro Ala Gly Ala Ile

100 105 110

Asp Ala Phe Asp Ser Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 24

<211> 110

<212> PRT

<213> Artificial

<220>

<223> light chain variable domain VL 1G9

<400> 24

Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Ser Glu Pro Val Gly

1 5 10 15

Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Glu Ser Ile Ser Asn Tyr

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly

50 55 60

Ser Gly Ser Gly Thr Glu Tyr Thr Val Thr Ile Ser Gly Val Gln Ser

65 70 75 80

Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ser Ser Asn

85 90 95

Val Phe Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val Lys

100 105 110

<210> 25

<211> 6

<212> PRT

<213> Artificial

<220>

<223> heavy chain CDR-H12F 6

<400> 25

Asn Asn Tyr Tyr Met Cys

1 5

<210> 26

<211> 17

<212> PRT

<213> Artificial

<220>

<223> heavy chain CDR-H22F 6

<400> 26

Cys Ile Ser Thr Asp Asp Ser Asn Thr His Tyr Ala Ser Trp Ala Gln

1 5 10 15

Gly

<210> 27

<211> 13

<212> PRT

<213> Artificial

<220>

<223> heavy chain CDR-H32F 6

<400> 27

Asp Ala His Phe Thr Ser Tyr Gly Tyr Gly Phe Asp Leu

1 5 10

<210> 28

<211> 11

<212> PRT

<213> Artificial

<220>

<223> light chain CDR-L12F 6

<400> 28

Arg Ala Ser Glu Asp Ile Glu Asn Leu Val Ala

1 5 10

<210> 29

<211> 7

<212> PRT

<213> Artificial

<220>

<223> light chain CDR-L22F 6

<400> 29

Gln Ala Ser Lys Leu Ala Ser

1 5

<210> 30

<211> 12

<212> PRT

<213> Artificial

<220>

<223> light chain CDR-L32F 6

<400> 30

Gln Gly Asp Tyr Gly Ser Gly Trp Ile Met Tyr Ser

1 5 10

<210> 31

<211> 121

<212> PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH 2F6

<400> 31

Gln Ser Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Glu Gly Ser

1 5 10 15

Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Asn Asn Asn Tyr

20 25 30

Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Cys Ile Ser Thr Asp Asp Ser Asn Thr His Tyr Ala Ser Trp Ala

50 55 60

Gln Gly Arg Phe Thr Ile Ser Lys Ala Ser Ser Thr Ala Leu Thr Leu

65 70 75 80

Gln Val Ala Gly Leu Thr Val Ala Asp Met Ala Thr Tyr Phe Cys Ala

85 90 95

Arg Asp Ala His Phe Thr Ser Tyr Gly Tyr Gly Phe Asp Leu Trp Gly

100 105 110

Pro Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 32

<211> 110

<212> PRT

<213> Artificial

<220>

<223> light chain variable domain VL 2F6

<400> 32

Asp Ile Val Met Thr Gln Thr Pro Ala Ser Val Ser Ala Ala Val Gly

1 5 10 15

Gly Thr Val Ser Ile Asn Cys Arg Ala Ser Glu Asp Ile Glu Asn Leu

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Tyr Gln Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly

50 55 60

Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Gly Asp Leu Glu Ser

65 70 75 80

Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gly Asp Tyr Gly Ser Gly Trp

85 90 95

Ile Met Tyr Ser Phe Gly Gly Gly Thr Asp Leu Val Val Lys

100 105 110

<210> 33

<211> 5

<212> PRT

<213> Artificial

<220>

<223> heavy chain CDR-H1 CNTO888

<400> 33

Ser Tyr Gly Ile Ser

1 5

<210> 34

<211> 17

<212> PRT

<213> Artificial

<220>

<223> heavy chain CDR-H2 CNTO888

<400> 34

Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 35

<211> 10

<212> PRT

<213> Artificial

<220>

<223> heavy chain CDR-H3 CNTO888

<400> 35

Tyr Asp Gly Ile Tyr Gly Glu Leu Asp Phe

1 5 10

<210> 36

<211> 12

<212> PRT

<213> Artificial

<220>

<223> light chain CDR-L1 CNTO888

<400> 36

Arg Ala Ser Gln Ser Val Ser Asp Ala Tyr Leu Ala

1 5 10

<210> 37

<211> 7

<212> PRT

<213> Artificial

<220>

<223> light chain CDR-L2 CNTO888

<400> 37

Asp Ala Ser Ser Arg Ala Thr

1 5

<210> 38

<211> 10

<212> PRT

<213> Artificial

<220>

<223> light chain CDR-L3 CNTO888

<400> 38

His Gln Tyr Ile Gln Leu His Ser Phe Thr

1 5 10

<210> 39

<211> 119

<212> PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH CNTO888

<400> 39

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Gly Ile Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 40

<211> 109

<212> PRT

<213> Artificial

<220>

<223> light chain variable domain VL CNTO888

<400> 40

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Ser Arg Ala Thr Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile Gln Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 41

<211> 5

<212> PRT

<213> Artificial

<220>

<223> heavy chain CDR-H1 humanized 11K2 (= 11K2)

<400> 41

Asp Thr Tyr Met His

1 5

<210> 42

<211> 17

<212> PRT

<213> Artificial

<220>

<223> heavy chain CDR-H2 humanized 11K2 (= 11K2)

<400> 42

Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Phe Asp Pro Lys Phe Gln

1 5 10 15

Gly

<210> 43

<211> 8

<212> PRT

<213> Artificial

<220>

<223> heavy chain CDR-H3 humanized 11K2 (= 11K2)

<400> 43

Gly Val Phe Gly Phe Phe Asp Tyr

1 5

<210> 44

<211> 11

<212> PRT

<213> Artificial

<220>

<223> light chain CDR-L1 humanized 11K2 (= 11K2)

<400> 44

Lys Ala Thr Glu Asp Ile Tyr Asn Arg Leu Ala

1 5 10

<210> 45

<211> 7

<212> PRT

<213> Artificial

<220>

<223> light chain CDR-L2 humanized 11K2 (= 11K2)

<400> 45

Gly Ala Thr Ser Leu Glu Thr

1 5

<210> 46

<211> 9

<212> PRT

<213> Artificial

<220>

<223> light chain CDR-L3 humanized 11K2 (= 11K2)

<400> 46

Gln Gln Phe Trp Ser Ala Pro Tyr Thr

1 5

<210> 47

<211> 117

<212> PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH humanization 11K2 (= 11K2)

<400> 47

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser Asp Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 48

<211> 107

<212> PRT

<213> Artificial

<220>

<223> light chain variable domain VL humanized 11K2 (= 11K2)

<400> 48

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Thr Glu Asp Ile Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 49

<211> 5

<212> PRT

<213> Artificial

<220>

<223> heavy chain CDR-H1 ABN912

<400> 49

His Tyr Trp Met Ser

1 5

<210> 50

<211> 17

<212> PRT

<213> Artificial

<220>

<223> heavy chain CDR-H2 ABN912

<400> 50

Asn Ile Glu Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val Lys

1 5 10 15

Gly

<210> 51

<211> 13

<212> PRT

<213> Artificial

<220>

<223> heavy chain CDR-H3 ABN912

<400> 51

Asp Leu Glu Gly Leu His Gly Asp Gly Tyr Phe Asp Leu

1 5 10

<210> 52

<211> 11

<212> PRT

<213> Artificial

<220>

<223> light chain CDR-L1 ABN912

<400> 52

Arg Ala Ser Gln Gly Val Ser Ser Ala Leu Ala

1 5 10

<210> 53

<211> 7

<212> PRT

<213> Artificial

<220>

<223> light chain CDR-L2 ABN912

<400> 53

Asp Ala Ser Ser Leu Glu Ser

1 5

<210> 54

<211> 9

<212> PRT

<213> Artificial

<220>

<223> light chain CDR-L3 ABN912

<400> 54

Gln Gln Phe Asn Ser Tyr Pro Leu Thr

1 5

<210> 55

<211> 122

<212> PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH ABN912

<400> 55

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ala Asn Ile Glu Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Asp Leu Glu Gly Leu His Gly Asp Gly Tyr Phe Asp Leu Trp

100 105 110

Gly Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 56

<211> 107

<212> PRT

<213> Artificial

<220>

<223> light chain variable domain VL ABN912

<400> 56

Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ile Leu Thr Cys Arg Ala Ser Gln Gly Val Ser Ser Ala

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Pro Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Phe Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 57

<211> 6

<212> PRT

<213> Artificial

<220>

<223> heavy chain CDR-H1 mutant variant CNTO888

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> X is I or T

<400> 57

Ser His Tyr Gly Xaa Ser

1 5

<210> 58

<211> 17

<212> PRT

<213> Artificial

<220>

<223> heavy chain CDR-H2 mutant variant CNTO888

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X is V or I or H

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> X is P or H

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> X is H or G

<400> 58

Gly Xaa Ile Xaa Ile Phe Xaa Thr Ala Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 59

<211> 10

<212> PRT

<213> Artificial

<220>

<223> heavy chain CDR-H3 mutant variant CNTO888

<400> 59

Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe

1 5 10

<210> 60

<211> 12

<212> PRT

<213> Artificial

<220>

<223> light chain CDR-L1 mutant variant CNTO888

<400> 60

Arg Ala Ser Gln His Val Ser Asp Ala Tyr Leu Ala

1 5 10

<210> 61

<211> 7

<212> PRT

<213> Artificial

<220>

<223> light chain CDR-L2 mutant variant CNTO888

<400> 61

Asp Ala Ser Asp Arg Ala Glu

1 5

<210> 62

<211> 10

<212> PRT

<213> Artificial

<220>

<223> light chain CDR-L3 mutant variant CNTO888

<400> 62

His Gln Tyr Ile His Leu His Ser Phe Thr

1 5 10

<210> 63

<211> 29

<212> PRT

<213> Artificial

<220>

<223> heavy chain FR-H1 mutant variant CNTO888

<400> 63

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe

20 25

<210> 64

<211> 14

<212> PRT

<213> Artificial

<220>

<223> heavy chain FR-H2 mutant variant CNTO888

<400> 64

Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

1 5 10

<210> 65

<211> 32

<212> PRT

<213> Artificial

<220>

<223> heavy chain FR-H3 mutant variant CNTO888

<400> 65

Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met Glu

1 5 10 15

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 66

<211> 11

<212> PRT

<213> Artificial

<220>

<223> heavy chain FR-H4 mutant variant CNTO888

<400> 66

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 67

<211> 23

<212> PRT

<213> Artificial

<220>

<223> light chain FR-L1 mutant CNTO888

<400> 67

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys

20

<210> 68

<211> 15

<212> PRT

<213> Artificial

<220>

<223> light chain FR-L2 mutant CNTO888

<400> 68

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

1 5 10 15

<210> 69

<211> 32

<212> PRT

<213> Artificial

<220>

<223> light chain FR-L3 mutant CNTO888

<400> 69

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys

20 25 30

<210> 70

<211> 9

<212> PRT

<213> Artificial

<220>

<223> light chain FR-L4 mutant CNTO888

<400> 70

Gly Gln Gly Thr Lys Val Glu Ile Lys

1 5

<210> 71

<211> 119

<212> PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH mutant variant CNTO 888H 0695

<400> 71

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 72

<211> 119

<212> PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH mutant variant CNTO 888H 0625

<400> 72

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile His Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 73

<211> 119

<212> PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH mutant variant CNTO 888H 0634

<400> 73

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Thr Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile His Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 74

<211> 119

<212> PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH mutant variant CNTO 888H 0635

<400> 74

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly His Ile His Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 75

<211> 109

<212> PRT

<213> Artificial

<220>

<223> light chain variable domain VL mutant CNTO 888L 0616

<400> 75

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 76

<211> 5

<212> PRT

<213> Artificial

<220>

<223> heavy chain CDR-H1 mutant variants humanized 11K2

<400> 76

His Thr Tyr Met His

1 5

<210> 77

<211> 17

<212> PRT

<213> Artificial

<220>

<223> heavy chain CDR-H2 mutant variants humanized 11K2

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> X is D or E

<400> 77

Arg Ile Asp Pro Xaa Asn His Asn Thr Lys Phe Asp Pro Lys Phe Gln

1 5 10 15

Gly

<210> 78

<211> 8

<212> PRT

<213> Artificial

<220>

<223> heavy chain CDR-H3 mutant variants humanized 11K2

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> X is D or E

<400> 78

Gly Val Phe Gly Phe Phe Xaa His

1 5

<210> 79

<211> 11

<212> PRT

<213> Artificial

<220>

<223> light chain CDR-L1 mutant variants humanized 11K2

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> X is F or T

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> X is R or L

<400> 79

Lys Ala Xaa Glu Asp Ile Tyr Asn Arg Xaa Ala

1 5 10

<210> 80

<211> 7

<212> PRT

<213> Artificial

<220>

<223> light chain CDR-L2 mutant variants humanized 11K2

<400> 80

Gly Ala Thr Ser Leu Glu His

1 5

<210> 81

<211> 9

<212> PRT

<213> Artificial

<220>

<223> light chain CDR-L3 mutant variants humanized 11K2

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> X is W or R

<400> 81

Gln Gln Phe Xaa Ser Ala Pro Tyr Thr

1 5

<210> 82

<211> 30

<212> PRT

<213> Artificial

<220>

<223> heavy chain FR-H1 mutant variants humanized 11K2

<400> 82

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser

20 25 30

<210> 83

<211> 14

<212> PRT

<213> Artificial

<220>

<223> heavy chain FR-H2 mutant variant humanized 11K2

<400> 83

Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

1 5 10

<210> 84

<211> 32

<212> PRT

<213> Artificial

<220>

<223> heavy chain FR-H3 mutant variant humanized 11K2

<400> 84

Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu

1 5 10 15

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 85

<211> 11

<212> PRT

<213> Artificial

<220>

<223> heavy chain FR-H4 mutant variant humanized 11K2

<400> 85

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

1 5 10

<210> 86

<211> 23

<212> PRT

<213> Artificial

<220>

<223> light chain FR-L1 mutant variant 11K2

<400> 86

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys

20

<210> 87

<211> 15

<212> PRT

<213> Artificial

<220>

<223> light chain FR-L2 mutant variant humanized 11K2

<400> 87

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile His

1 5 10 15

<210> 88

<211> 32

<212> PRT

<213> Artificial

<220>

<223> light chain FR-L3 mutant variant humanized 11K2

<400> 88

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 89

<211> 10

<212> PRT

<213> Artificial

<220>

<223> light chain FR-L4 mutant variant humanized 11K2

<400> 89

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

1 5 10

<210> 90

<211> 117

<212> PRT

<213> Artificial

<220>

<223> humanization of heavy chain variable domain VH mutant variants 11K2

H1503

<400> 90

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Asp His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 91

<211> 117

<212> PRT

<213> Artificial

<220>

<223> humanization of heavy chain variable domain VH mutant variants 11K2

H1510

<400> 91

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Glu His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 92

<211> 117

<212> PRT

<213> Artificial

<220>

<223> humanization of heavy chain variable domain VH mutant variants 11K2

H1514

<400> 92

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Glu Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Glu His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 93

<211> 107

<212> PRT

<213> Artificial

<220>

<223> light chain variable domain VL mutant variants humanized 11K2

L1338

<400> 93

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Phe Glu Asp Ile Tyr Asn Arg

20 25 30

Arg Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 94

<211> 107

<212> PRT

<213> Artificial

<220>

<223> light chain variable domain VL mutant variants humanized 11K2

L1201

<400> 94

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Thr Glu Asp Ile Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Arg Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 95

<211> 107

<212> PRT

<213> Intelligent people

<400> 95

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 96

<211> 105

<212> PRT

<213> Intelligent people

<400> 96

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

1 5 10 15

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

20 25 30

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

35 40 45

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

50 55 60

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

65 70 75 80

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

85 90 95

Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210> 97

<211> 328

<212> PRT

<213> Intelligent people

<400> 97

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 98

<211> 328

<212> PRT

<213> Artificial

<220>

<223> exemplary human heavy chain constant region derived from IgG1 with mutations L234A, L235A, and P329G (Fc gamma receptor silencing)

<400> 98

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 99

<211> 328

<212> PRT

<213> Intelligent people

<400> 99

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 100

<211> 328

<212> PRT

<213> Artificial

<220>

<223> exemplary human heavy chain constant region derived from IgG1 with mutation (SG 105-IgG1 allotype-Fc γ receptor silencing)

<400> 100

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Arg Arg Gly Pro Lys Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 101

<211> 328

<212> PRT

<213> Artificial

<220>

<223> SG 1095-exemplary human heavy chain constant region derived from IgG1 including mutations (Kabat EU numbering)

L235W/G236N/H268D/Q295L/A330K/K326T, Q311R/P343R, N434A,

Q438R/S440E

<400> 101

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Trp Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Leu Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Thr Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 102

<211> 328

<212> PRT

<213> Artificial

<220>

<223> SG 1099-exemplary human heavy chain constant region derived from IgG1 including the mutation (Kabat EU numbering) Q311R/P343R

<400> 102

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 103

<211> 328

<212> PRT

<213> Artificial

<220>

<223> SG 1100-an exemplary human heavy chain constant region derived from IgG1 including mutations (Kabat EU numbering) Q311R/P343R, N434A, Q438R/S440E

<400> 103

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 104

<211> 437

<212> PRT

<213> Artificial

<220>

<223> heavy chain 1-CNTO 888//11K2-WT IgG1

<400> 104

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Thr Glu Asp Ile Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

420 425 430

Leu Ser Leu Ser Pro

435

<210> 105

<211> 447

<212> PRT

<213> Artificial

<220>

<223> heavy chain 2-CNTO 888//11K2-WT IgG1

<400> 105

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Gly Ile Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 106

<211> 224

<212> PRT

<213> Artificial

<220>

<223> light chain 1-CNTO 888//11K2-WT IgG1

<400> 106

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser Asp Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 107

<211> 216

<212> PRT

<213> Artificial

<220>

<223> light chain 2-CNTO 888//11K2-WT IgG1

<400> 107

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Ser Arg Ala Thr Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile Gln Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 108

<211> 437

<212> PRT

<213> Artificial

<220>

<223> heavy chain 1-CKLO 2 IgG1

<400> 108

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Phe Glu Asp Ile Tyr Asn Arg

20 25 30

Arg Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

420 425 430

Leu Ser Leu Ser Pro

435

<210> 109

<211> 447

<212> PRT

<213> Artificial

<220>

<223> heavy chain 2-CKLO 2 IgG1

<400> 109

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 110

<211> 224

<212> PRT

<213> Artificial

<220>

<223> light chain 1-CKLO 2 IgG1

<400> 110

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Glu His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 111

<211> 216

<212> PRT

<213> Artificial

<220>

<223> light chain 2-CKLO 2 IgG1

<400> 111

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 112

<211> 437

<212> PRT

<213> Artificial

<220>

<223> heavy chain 1-CKLO 2-SG1095

<400> 112

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Phe Glu Asp Ile Tyr Asn Arg

20 25 30

Arg Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Leu Trp Asn Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

245 250 255

Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Leu Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Arg Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Thr Ala Leu

305 310 315 320

Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Arg Lys Glu

420 425 430

Leu Ser Leu Ser Pro

435

<210> 113

<211> 447

<212> PRT

<213> Artificial

<220>

<223> heavy chain 2-CKLO 2-SG1095

<400> 113

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Trp Asn Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Asp Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Leu Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Arg Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Thr Ala Leu Pro Lys Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Ala His Tyr Thr Arg Lys Glu Leu Ser Leu Ser Pro

435 440 445

<210> 114

<211> 224

<212> PRT

<213> Artificial

<220>

<223> light chain 1-CKLO 2-SG1095

<400> 114

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Glu His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 115

<211> 216

<212> PRT

<213> Artificial

<220>

<223> light chain 2-CKLO 2-SG1095

<400> 115

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 116

<211> 437

<212> PRT

<213> Artificial

<220>

<223> heavy chain 1-CKLO 2-SG1099

<400> 116

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Phe Glu Asp Ile Tyr Asn Arg

20 25 30

Arg Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Arg Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

420 425 430

Leu Ser Leu Ser Pro

435

<210> 117

<211> 447

<212> PRT

<213> Artificial

<220>

<223> heavy chain 2-CKLO 2-SG1099

<400> 117

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Arg Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 118

<211> 224

<212> PRT

<213> Artificial

<220>

<223> light chain 1-CKLO 2-SG1099

<400> 118

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Glu His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 119

<211> 216

<212> PRT

<213> Artificial

<220>

<223> light chain 2-CKLO 2-SG1099

<400> 119

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 120

<211> 437

<212> PRT

<213> Artificial

<220>

<223> heavy chain 1-CKLO 2-SG1100

<400> 120

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Phe Glu Asp Ile Tyr Asn Arg

20 25 30

Arg Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Arg Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Arg Lys Glu

420 425 430

Leu Ser Leu Ser Pro

435

<210> 121

<211> 447

<212> PRT

<213> Artificial

<220>

<223> heavy chain 2-CKLO 2-SG1100

<400> 121

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Arg Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Ala His Tyr Thr Arg Lys Glu Leu Ser Leu Ser Pro

435 440 445

<210> 122

<211> 224

<212> PRT

<213> Artificial

<220>

<223> light chain 1-CKLO 2-SG1100

<400> 122

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Glu His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 123

<211> 216

<212> PRT

<213> Artificial

<220>

<223> light chain 2-CKLO 2-SG1100

<400> 123

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 124

<211> 437

<212> PRT

<213> Artificial

<220>

<223> heavy chain 1-CKLO 3-SG1095

<400> 124

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Thr Glu Asp Ile Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Arg Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Leu Trp Asn Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

245 250 255

Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Leu Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Arg Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Thr Ala Leu

305 310 315 320

Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Arg Lys Glu

420 425 430

Leu Ser Leu Ser Pro

435

<210> 125

<211> 447

<212> PRT

<213> Artificial

<220>

<223> heavy chain 2-CKLO 3-SG1095

<400> 125

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Trp Asn Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Asp Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Leu Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Arg Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Thr Ala Leu Pro Lys Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Ala His Tyr Thr Arg Lys Glu Leu Ser Leu Ser Pro

435 440 445

<210> 126

<211> 224

<212> PRT

<213> Artificial

<220>

<223> light chain 1-CKLO 3-SG1095

<400> 126

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Asp His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 127

<211> 216

<212> PRT

<213> Artificial

<220>

<223> light chain 2-CKLO 3-SG1095

<400> 127

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 128

<211> 437

<212> PRT

<213> Artificial

<220>

<223> heavy chain 1-CKLO 3-SG1099

<400> 128

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Thr Glu Asp Ile Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Arg Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Arg Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

420 425 430

Leu Ser Leu Ser Pro

435

<210> 129

<211> 447

<212> PRT

<213> Artificial

<220>

<223> heavy chain 2-CKLO 3-SG1099

<400> 129

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Arg Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 130

<211> 224

<212> PRT

<213> Artificial

<220>

<223> light chain 1-CKLO 3-SG1099

<400> 130

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Asp His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 131

<211> 216

<212> PRT

<213> Artificial

<220>

<223> light chain 2-CKLO 3-SG1099

<400> 131

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 132

<211> 437

<212> PRT

<213> Artificial

<220>

<223> heavy chain 1-CKLO 3-SG1100

<400> 132

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Thr Glu Asp Ile Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Arg Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Arg Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Arg Lys Glu

420 425 430

Leu Ser Leu Ser Pro

435

<210> 133

<211> 447

<212> PRT

<213> Artificial

<220>

<223> heavy chain 2-CKLO 3-SG1100

<400> 133

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Arg Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Ala His Tyr Thr Arg Lys Glu Leu Ser Leu Ser Pro

435 440 445

<210> 134

<211> 224

<212> PRT

<213> Artificial

<220>

<223> light chain 1-CKLO 3-SG1100

<400> 134

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Asp His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 135

<211> 216

<212> PRT

<213> Artificial

<220>

<223> light chain 2-CKLO 3-SG1100

<400> 135

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 136

<211> 115

<212> PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH 2F2

<400> 136

Gln Glu Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Thr Leu Thr Leu Thr Cys Lys Ala Ser Gly Phe Ser Phe Ile Asp His

20 25 30

Tyr Ala Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Ala Tyr Ile Gly Gly Ser Gly Thr Thr Tyr Tyr Ala Asn Arg Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Thr Ser Ser Thr Thr Val Thr Leu Gln

65 70 75 80

Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala Arg

85 90 95

Asn Leu Asp Val Ser Ala Ser Leu Trp Gly Pro Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 137

<211> 112

<212> PRT

<213> Artificial

<220>

<223> light chain variable domain VL 2F2

<400> 137

Gln Val Leu Thr Gln Thr Pro Ala Ser Val Ser Ala Ala Val Gly Gly

1 5 10 15

Thr Val Thr Ile Ser Cys Gln Ser Ser Gln Ser Val Tyr Asn Asn Asn

20 25 30

Leu Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu

35 40 45

Ile Tyr Glu Ala Ser Lys Leu Ala Ser Gly Val Pro Pro Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Gln Ser Thr Leu Thr Ile Ser Gly Val Gln

65 70 75 80

Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Glu Gly Ala Phe Leu Cys Thr

85 90 95

Thr Tyr Asp Cys Phe Val Phe Gly Gly Gly Thr Glu Val Val Val Lys

100 105 110

<210> 138

<211> 117

<212> PRT

<213> mice

<400> 138

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Ala Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Pro Ala Ser Gly Leu Asn Ile Lys Asp Thr

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210> 139

<211> 107

<212> PRT

<213> mice

<400> 139

Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Val Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Thr Glu Asp Ile Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Ser Ala Pro Arg Leu Leu Ile

35 40 45

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Thr Ser Leu Gln Thr

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 140

<211> 121

<212> PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH 1H11

<400> 140

Gln Glu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Glu Gly

1 5 10 15

Ser Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Ser Thr Ser

20 25 30

Tyr Trp Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu

35 40 45

Ile Ala Cys Ile Ser Ser Ser Ile Gly Val Thr Tyr Tyr Ala Ser Trp

50 55 60

Ala Glu Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Thr

65 70 75 80

Leu Gln Met Thr Ser Leu Thr Val Ala Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Thr Thr Asp Asp Asn Trp Asn Val Gly Phe Asn Leu Trp Gly

100 105 110

Pro Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 141

<211> 110

<212> PRT

<213> Artificial

<220>

<223> light chain variable domain VL 1H11

<400> 141

Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Gly Val Gly Gly

1 5 10 15

Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Gly Asn Arg Tyr

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Thr Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly

50 55 60

Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Glu Cys

65 70 75 80

Ala Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Gly Ala Thr Ile Ser Tyr

85 90 95

Leu Asp Asn Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys

100 105 110

<210> 142

<211> 76

<212> PRT

<213> Intelligent people

<400> 142

Gln Pro Asp Ala Ile Asn Ala Pro Val Thr Cys Cys Tyr Asn Phe Thr

1 5 10 15

Asn Arg Lys Ile Ser Val Gln Arg Leu Ala Ser Tyr Arg Arg Ile Thr

20 25 30

Ser Ser Lys Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Ile Val Ala

35 40 45

Lys Glu Ile Cys Ala Asp Pro Lys Gln Lys Trp Val Gln Asp Ser Met

50 55 60

Asp His Leu Asp Lys Gln Thr Gln Thr Pro Lys Thr

65 70 75

<210> 143

<211> 76

<212> PRT

<213> Artificial

<220>

<223> exemplary human CCL2 (MCP1) -P8A variants

<400> 143

Gln Pro Asp Ala Ile Asn Ala Ala Val Thr Cys Cys Tyr Asn Phe Thr

1 5 10 15

Asn Arg Lys Ile Ser Val Gln Arg Leu Ala Ser Tyr Arg Arg Ile Thr

20 25 30

Ser Ser Lys Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Ile Val Ala

35 40 45

Lys Glu Ile Cys Ala Asp Pro Lys Gln Lys Trp Val Gln Asp Ser Met

50 55 60

Asp His Leu Asp Lys Gln Thr Gln Thr Pro Lys Thr

65 70 75

<210> 144

<211> 76

<212> PRT

<213> Artificial

<220>

<223> exemplary human CCL2 (MCP1) -T10C variants

<400> 144

Gln Pro Asp Ala Ile Asn Ala Pro Val Cys Cys Cys Tyr Asn Phe Thr

1 5 10 15

Asn Arg Lys Ile Ser Val Gln Arg Leu Ala Ser Tyr Arg Arg Ile Thr

20 25 30

Ser Ser Lys Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Ile Val Ala

35 40 45

Lys Glu Ile Cys Ala Asp Pro Lys Gln Lys Trp Val Gln Asp Ser Met

50 55 60

Asp His Leu Asp Lys Gln Thr Gln Thr Pro Lys Thr

65 70 75

<210> 145

<211> 76

<212> PRT

<213> Intelligent people

<400> 145

Gln Pro Asp Ser Val Ser Ile Pro Ile Thr Cys Cys Phe Asn Val Ile

1 5 10 15

Asn Arg Lys Ile Pro Ile Gln Arg Leu Glu Ser Tyr Thr Arg Ile Thr

20 25 30

Asn Ile Gln Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Lys Arg Gly

35 40 45

Lys Glu Val Cys Ala Asp Pro Lys Glu Arg Trp Val Arg Asp Ser Met

50 55 60

Lys His Leu Asp Gln Ile Phe Gln Asn Leu Lys Pro

65 70 75

<210> 146

<211> 76

<212> PRT

<213> Artificial

<220>

<223> SEQ ID NO: 146 exemplary human CCL8 (MCP2) -P8A variant

<400> 146

Gln Pro Asp Ser Val Ser Ile Ala Ile Thr Cys Cys Phe Asn Val Ile

1 5 10 15

Asn Arg Lys Ile Pro Ile Gln Arg Leu Glu Ser Tyr Thr Arg Ile Thr

20 25 30

Asn Ile Gln Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Lys Arg Gly

35 40 45

Lys Glu Val Cys Ala Asp Pro Lys Glu Arg Trp Val Arg Asp Ser Met

50 55 60

Lys His Leu Asp Gln Ile Phe Gln Asn Leu Lys Pro

65 70 75

<210> 147

<211> 76

<212> PRT

<213> Intelligent people

<400> 147

Gln Pro Val Gly Ile Asn Thr Ser Thr Thr Cys Cys Tyr Arg Phe Ile

1 5 10 15

Asn Lys Lys Ile Pro Lys Gln Arg Leu Glu Ser Tyr Arg Arg Thr Thr

20 25 30

Ser Ser His Cys Pro Arg Glu Ala Val Ile Phe Lys Thr Lys Leu Asp

35 40 45

Lys Glu Ile Cys Ala Asp Pro Thr Gln Lys Trp Val Gln Asp Phe Met

50 55 60

Lys His Leu Asp Lys Lys Thr Gln Thr Pro Lys Leu

65 70 75

<210> 148

<211> 82

<212> PRT

<213> Intelligent people

<400> 148

Phe Asn Pro Gln Gly Leu Ala Gln Pro Asp Ala Leu Asn Val Pro Ser

1 5 10 15

Thr Cys Cys Phe Thr Phe Ser Ser Lys Lys Ile Ser Leu Gln Arg Leu

20 25 30

Lys Ser Tyr Val Ile Thr Thr Ser Arg Cys Pro Gln Lys Ala Val Ile

35 40 45

Phe Arg Thr Lys Leu Gly Lys Glu Ile Cys Ala Asp Pro Lys Glu Lys

50 55 60

Trp Val Gln Asn Tyr Met Lys His Leu Gly Arg Lys Ala His Thr Leu

65 70 75 80

Lys Thr

<210> 149

<211> 76

<212> PRT

<213> cynomolgus monkey

<400> 149

Gln Pro Asp Ala Ile Asn Ala Pro Val Thr Cys Cys Tyr Asn Phe Thr

1 5 10 15

Asn Arg Lys Ile Ser Val Gln Arg Leu Ala Ser Tyr Arg Arg Ile Thr

20 25 30

Ser Ser Lys Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Ile Val Ala

35 40 45

Lys Glu Ile Cys Ala Asp Pro Lys Gln Lys Trp Val Gln Asp Ser Met

50 55 60

Asp His Leu Asp Lys Gln Ile Gln Thr Pro Lys Pro

65 70 75

<210> 150

<211> 125

<212> PRT

<213> mice

<400> 150

Gln Pro Asp Ala Val Asn Ala Pro Leu Thr Cys Cys Tyr Ser Phe Thr

1 5 10 15

Ser Lys Met Ile Pro Met Ser Arg Leu Glu Ser Tyr Lys Arg Ile Thr

20 25 30

Ser Ser Arg Cys Pro Lys Glu Ala Val Val Phe Val Thr Lys Leu Lys

35 40 45

Arg Glu Val Cys Ala Asp Pro Lys Lys Glu Trp Val Gln Thr Tyr Ile

50 55 60

Lys Asn Leu Asp Arg Asn Gln Met Arg Ser Glu Pro Thr Thr Leu Phe

65 70 75 80

Lys Thr Ala Ser Ala Leu Arg Ser Ser Ala Pro Leu Asn Val Lys Leu

85 90 95

Thr Arg Lys Ser Glu Ala Asn Ala Ser Thr Thr Phe Ser Thr Thr Thr

100 105 110

Ser Ser Thr Ser Val Gly Val Thr Ser Val Thr Val Asn

115 120 125

<210> 151

<211> 328

<212> PRT

<213> Artificial

<220>

<223> GG 01-exemplary human heavy chain constant region derived from IgG1 including mutations (Kabat EU numbering)

L234Y/P238D/T250V/V264I/T307P/A330K, Q311R/P343R, N434A,

Q438R/S440E

<400> 151

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Tyr Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Ile Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu

180 185 190

His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 152

<211> 328

<212> PRT

<213> Artificial

<220>

<223> GG 02-exemplary human heavy chain constant region derived from IgG1 including mutations (Kabat EU numbering)

L234Y/P238D/T250V/V264I/T307P/A330K, Q311R/P343R,

M428L/N434A/Y436T,Q438R/S440E

<400> 152

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Tyr Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Ile Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu

180 185 190

His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ala His Thr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 153

<211> 328

<212> PRT

<213> Artificial

<220>

<223> GG 03-derived from an exemplary human heavy chain constant region (Kabat EU numbering) L234Y/P238D/T250V/V264I/T307P/A330K, Q311R/P343R, N434A, Q438R/S440E including mutated IgG1 (comprising an IgG1 allotype sequence)

<400> 153

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Tyr Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Ile Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu

180 185 190

His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 154

<211> 328

<212> PRT

<213> Artificial

<220>

<223> GG 04-exemplary human heavy chain constant region derived from IgG1 (comprising IgG1 allotype sequence) including mutations (Kabat EU numbering) L234Y/P238D/T250V/V264I/T307P/A330K, Q311R/P343R, M428L/N434A/Y436T, Q438R/S440E

<400> 154

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Tyr Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Ile Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu

180 185 190

His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ala His Thr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 155

<211> 437

<212> PRT

<213> Artificial

<220>

<223> heavy chain 1-CKLO 2-GG01

<400> 155

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Phe Glu Asp Ile Tyr Asn Arg

20 25 30

Arg Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Tyr Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Ile

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu His Arg Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Arg Lys Glu

420 425 430

Leu Ser Leu Ser Pro

435

<210> 156

<211> 447

<212> PRT

<213> Artificial

<220>

<223> heavy chain 2-CKLO 2-GG01

<400> 156

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Tyr Leu Gly Gly Asp

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Ile Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Pro Val Leu His Arg Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Lys Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Ala His Tyr Thr Arg Lys Glu Leu Ser Leu Ser Pro

435 440 445

<210> 157

<211> 224

<212> PRT

<213> Artificial

<220>

<223> light chain 1-CKLO 2 GG01

<400> 157

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Glu His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 158

<211> 216

<212> PRT

<213> Artificial

<220>

<223> light chain 2-CKLO 2 GG01

<400> 158

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 159

<211> 437

<212> PRT

<213> Artificial

<220>

<223> heavy chain 1-CKLO 2 GG02

<400> 159

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Phe Glu Asp Ile Tyr Asn Arg

20 25 30

Arg Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Tyr Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Ile

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu His Arg Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Leu His Glu Ala Leu His Ala His Thr Thr Arg Lys Glu

420 425 430

Leu Ser Leu Ser Pro

435

<210> 160

<211> 447

<212> PRT

<213> Artificial

<220>

<223> heavy chain 2-CKLO 2 GG02

<400> 160

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Tyr Leu Gly Gly Asp

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Ile Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Pro Val Leu His Arg Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Lys Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu

420 425 430

Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu Ser Pro

435 440 445

<210> 161

<211> 224

<212> PRT

<213> Artificial

<220>

<223> light chain 1-CKLO 2-GG02

<400> 161

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Glu His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 162

<211> 216

<212> PRT

<213> Artificial

<220>

<223> light chain 2-CKLO 2-GG02

<400> 162

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 163

<211> 437

<212> PRT

<213> Artificial

<220>

<223> heavy chain 1-CKLO 2-GG03

<400> 163

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Phe Glu Asp Ile Tyr Asn Arg

20 25 30

Arg Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Tyr Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Ile

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu His Arg Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Arg Lys Glu

420 425 430

Leu Ser Leu Ser Pro

435

<210> 164

<211> 447

<212> PRT

<213> Artificial

<220>

<223> heavy chain 2-CKLO 2-GG03

<400> 164

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Tyr Leu Gly Gly Asp

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Ile Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Pro Val Leu His Arg Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Lys Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Ala His Tyr Thr Arg Lys Glu Leu Ser Leu Ser Pro

435 440 445

<210> 165

<211> 224

<212> PRT

<213> Artificial

<220>

<223> light chain 1-CKLO 2-GG03

<400> 165

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Glu His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 166

<211> 216

<212> PRT

<213> Artificial

<220>

<223> light chain 2-CKLO 2-GG03

<400> 166

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 167

<211> 437

<212> PRT

<213> Artificial

<220>

<223> heavy chain 1-CKLO 2-GG04

<400> 167

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Phe Glu Asp Ile Tyr Asn Arg

20 25 30

Arg Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Tyr Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Ile

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu His Arg Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Leu His Glu Ala Leu His Ala His Thr Thr Arg Lys Glu

420 425 430

Leu Ser Leu Ser Pro

435

<210> 168

<211> 447

<212> PRT

<213> Artificial

<220>

<223> heavy chain 2-CKLO 2-GG04

<400> 168

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Tyr Leu Gly Gly Asp

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Ile Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Pro Val Leu His Arg Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Lys Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu

420 425 430

Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu Ser Pro

435 440 445

<210> 169

<211> 224

<212> PRT

<213> Artificial

<220>

<223> light chain 1-CKLO 2-GG04

<400> 169

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Glu His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 170

<211> 216

<212> PRT

<213> Artificial

<220>

<223> light chain 2-CKLO 2-GG04

<400> 170

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 171

<211> 437

<212> PRT

<213> Artificial

<220>

<223> heavy chain 1-CKLO 2-GG03/GG04

<400> 171

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Phe Glu Asp Ile Tyr Asn Arg

20 25 30

Arg Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Tyr Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Ile

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu His Arg Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Arg Lys Glu

420 425 430

Leu Ser Leu Ser Pro

435

<210> 172

<211> 447

<212> PRT

<213> Artificial

<220>

<223> heavy chain 2-CKLO 2-GG03/GG04

<400> 172

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Tyr Leu Gly Gly Asp

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Ile Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Pro Val Leu His Arg Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Lys Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu

420 425 430

Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu Ser Pro

435 440 445

<210> 173

<211> 224

<212> PRT

<213> Artificial

<220>

<223> light chain 1-CKLO 2-GG03/GG04

<400> 173

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Glu His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 174

<211> 216

<212> PRT

<213> Artificial

<220>

<223> light chain 2-CKLO 2-GG03/GG04

<400> 174

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

Claims

1. A bispecific antibody comprising a first antigen-binding site that binds 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 an Fc domain of a human IgG isotype.

2. The bispecific antibody according to claim 1,

wherein

A) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

B) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

C) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

A VH domain comprising: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO. 9; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO. 10; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO. 11;

or

D) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

E) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

Or alternatively

F) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

G) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

H) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

I) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

3. The bispecific antibody according to claim 2,

wherein

A) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 39, wherein the VH domain comprises: (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 said VL domain comprises: (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; and is

ii) the second antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 47, wherein the VH domain comprises: (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 said VL domain comprises: (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;

or

B) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

And a VL domain comprising the amino acid sequence of SEQ ID NO 24, wherein said VL domain comprises: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO. 20; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO 21; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO. 22;

or

C) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

And a VL domain comprising the amino acid sequence of SEQ ID NO:16, wherein said VL domain comprises: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO. 12; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO 13; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO. 14;

or

D) i) the first antigen binding site comprises

and a VL domain comprising the amino acid sequence of SEQ ID NO 24, wherein said VL domain comprises: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO. 20; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO 21; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO. 22; and is

ii) the second antigen binding site comprises

And a VL domain comprising the amino acid sequence of SEQ ID NO:48, wherein the VL domain comprises: (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;

or

E) i) the first antigen binding site comprises

and a VL domain comprising the amino acid sequence of SEQ ID NO:32, wherein said VL domain comprises: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO 28; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 29; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30; and is

ii) the second antigen binding site comprises

And a VL domain comprising the amino acid sequence of SEQ ID NO:48, wherein the VL domain comprises: (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;

or

F) i) the first antigen binding site comprises

and a VL domain comprising the amino acid sequence of SEQ ID NO 56, wherein said VL domain comprises: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO 52; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO 53; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; and is

ii) the second antigen binding site comprises

or

G) i) the first antigen binding site comprises

and a VL domain comprising the amino acid sequence of SEQ ID NO:16, wherein said VL domain comprises: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO. 12; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO 13; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO. 14; and is

ii) the second antigen binding site comprises

And a VL domain comprising the amino acid sequence of SEQ ID No. 24, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO. 20; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 21; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO. 22;

or

H) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

And a VL domain comprising the amino acid sequence of SEQ ID NO:32, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO 28; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 29; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30;

or

I) i) the first antigen binding site comprises

and a VL domain comprising the amino acid sequence of SEQ ID No. 8, wherein said VL domain comprises: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO. 4; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO 5; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO 6; and is

ii) the second antigen binding site comprises

And a VL domain comprising the amino acid sequence of SEQ ID NO:32, wherein said VL domain comprises: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO 28; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 29; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

4. The bispecific antibody of any one of claims 1 to 3, wherein the bispecific antibody comprising an Fc domain of human IgG isotype is a bispecific antibody comprising a constant heavy chain domain of human IgG1 isotype.

5. The bispecific antibody of claim 4, wherein the in vivo clearance (ml/day/kg) of human CCL2 is at least two-fold higher after administration of a bispecific antibody comprising a constant heavy chain domain of human wild-type IgG1 isotype compared to the in vivo clearance (ml/day/kg) of human CCL2 after administration of a bispecific antibody comprising an Fcyreceptor silencing constant heavy chain domain of human IgG1 isotype comprising mutations L234A, L235A, P329G ((Kabat EU numbering) when a preformed immune complex consisting of 20mg/kg of each bispecific antibody and 0.1mg/kg of human CCL2 is administered to an FcRn transgenic mouse in a single dose of 10 ml/kg.

6. 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,

Wherein

ii) the second antigen binding site binds to the same epitope on CCL2 as does an antibody comprising

and a VL domain comprising the amino acid sequence of SEQ ID NO:48, wherein said VL domain comprises: (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.

7. The bispecific antibody of claim 6, wherein the in vivo clearance (ml/day/kg) of human CCL2 is at least 15-fold higher, in particular at least 20-fold higher, after administration of a bispecific antibody comprising a constant heavy chain domain of human wild-type IgG1 isotype compared to the in vivo clearance (ml/day/kg) of human CCL2 after administration of a bispecific antibody comprising an Fcy receptor silencing constant heavy chain domain of human IgG1 isotype comprising mutations L234A, L235A, P329G ((Kabat EU numbering) when a preformed immune complex consisting of 20mg/kg of each bispecific antibody and 0.1mg/kg of human CCL2 is administered to an FcRn transgenic mouse at a single dose of 10 ml/kg.

wherein

i) The first antigen binding site comprises

and

and is

ii) the second antigen binding site comprises

and

a VL domain comprising: (d) CDR-L1 comprising the amino acid sequence KAX of SEQ ID NO:79 ¹ EDIYNRX ² A, wherein X ¹ Is F or T and X ² Is R or L; (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, wherein X is W or R.

Wherein

i) The first antigen binding site comprises

A VH domain comprising: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO 57, wherein X is I or T; (b) CDR-H2 comprising the amino acid sequence GX of SEQ ID NO:58 ¹ IX ² IFX ³ TANYAQKFQG, wherein X ¹ Is V, I or H, X ² Is P or H, and X ³ Is H or G; (c) CDR-H3 comprising amino acid sequence YDAHYGELDF of SEQ ID NO: 59; (d) FR-H1 comprising the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTF of SEQ ID NO: 63; (e) FR-H2 comprising the amino acid sequence WVRQAPGQGLEWMG of SEQ ID NO 64; (f) FR-H3 comprising the amino acid sequence RVTITADESTSTAYMELSSLRSEDTAVYYCAR of SEQ ID NO: 65; and (g) FR-H4 comprising the amino acid sequence WGQGTLVTVSS of SEQ ID NO: 66;

and

And is

ii) the second antigen binding site comprises

A VH domain 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, wherein X is D or E; (c) CDR-H3 comprising the amino acid sequence GVGFFXH of SEQ ID NO. 78, wherein X is D or E; (d) FR-H1 comprising the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGLTIS of SEQ ID NO: 82; (e) FR-H2 comprising the amino acid sequence WVRQAPGQGLEWMG of SEQ ID NO 83; (f) FR-H3 comprising the amino acid sequence RVTITADTSTSTAYMELSSLRSEDTAVYYCAR of SEQ ID NO: 84; and (g) FR-H4 comprising amino acid sequence WGQGTTVTVSS of SEQ ID NO: 85;

and

a VL domain comprising: (h) CDR-L1 comprising the amino acid sequence KAX of SEQ ID NO:79 ¹ EDIYNRX ² A, wherein X ¹ Is F or T and X ² Is R or L; (i) CDR-L2 comprising the amino acid sequence GATSLEH of SEQ ID NO. 80; (j) CDR-L3 comprising the amino acid sequence QQFXSAPYT of SEQ ID NO. 81, wherein X is W or R; (k) FR-L1 comprising the amino acid sequence DIQMTQSPSSLSASVGDRVTITC of SEQ ID NO 86; (l) FR-L2 comprising the amino acid sequence WYQQKPGKAPKLLIH of SEQ ID NO: 87; (m) FR-L3 comprising the amino acid sequence GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC of SEQ ID NO: 88; and (n) FR-L4 comprising the amino acid sequence FGGGTKVEIK of SEQ ID NO: 89.

wherein

A) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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

B) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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

C) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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 94;

or

D) 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 is

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 94;

or

E) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 73;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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

F) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 73;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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 94;

or

G) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 73;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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

H) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 73;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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

I) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 72;

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 alternatively

J) i) the first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO: 72;

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

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 is

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

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 is

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

M) i) said first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO: 74;

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 94;

or alternatively

N) 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 is

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

O) i) said 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 is

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

P) i) said first antigen binding site comprises

A VH domain comprising the amino acid sequence of SEQ ID NO 71;

And a VL domain comprising the amino acid sequence of SEQ ID NO 75; and is

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.

wherein

A) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO. 90, wherein the VH domain comprises: (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, wherein X is D; and (c) CDR-H3 comprising the amino acid sequence GVGFFXH of SEQ ID NO:78 wherein 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 to the amino acid sequence of SEQ ID NO:93, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence KAX of SEQ ID NO:79 ¹ EDIYNRX ² A, wherein X ¹ Is F and X ² Is R; (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, wherein X is W;

or

B) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

C) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID No. 94, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence KAX of SEQ ID NO:79 ¹ EDIYNRX ² A, wherein X ¹ Is F or T and X ² Is R or L; (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, wherein X is W or R;

or

D) i) the first antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO 72, wherein the VH domain comprises: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO 57, wherein X is I or T; (b) CDR-H2 comprising the amino acid sequence GX of SEQ ID NO:58 ¹ IX ² IFX ³ TANYAQKFQG, wherein X ¹ Is V, I or H, X ² Is P or H, and X ³ Is H or G; and (c) CDR-H3 comprising amino acid sequence YDAHYGELDF of SEQ ID NO: 59;

ii) the second antigen binding site comprises

or

E) i) the first antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO 73, wherein the VH domain comprises: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:57Column SHYGXS, where X is I or T; (b) CDR-H2 comprising the amino acid sequence GX of SEQ ID NO:58 ¹ IX ² IFX ³ TANYAQKFQG, wherein X ¹ Is V, I or H, X ² Is P or H, and X ³ Is H or G; and (c) CDR-H3 comprising amino acid sequence YDAHYGELDF of SEQ ID NO: 59;

ii) the second antigen binding site comprises

and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:93, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence KAX of SEQ ID NO:79 ¹ EDIYNRX ² A, wherein X ¹ Is F or T and X ² Is R or L; (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, wherein X is W or R;

or

F) i) the first antigen binding site comprises

VH domain sequence having at least 90%, 91%, 92%, 93% amino acid sequence of SEQ ID NO 73, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, wherein the VH domain comprises: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO 57, wherein X is I or T; (b) CDR-H2 comprising the amino acid sequence GX of SEQ ID NO:58 ¹ IX ² IFX ³ TANYAQKFQG, wherein X ¹ Is V, I or H, X ² Is P or H, and X ³ Is H or G; and (c) CDR-H3 comprising amino acid sequence YDAHYGELDF of SEQ ID NO: 59;

ii) the second antigen binding site comprises

or

G) i) the first antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO 73, wherein the VH domain comprises: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO 57, wherein X is I or T; (b) CDR-H2 comprising the amino acid sequence GX of SEQ ID NO:58 ¹ IX ² IFX ³ TANYAQKFQG, wherein X ¹ Is V, I or H, X ² Is P or H, and X ³ Is H or G; and (c) CDR-H3 comprising amino acid sequence YDAHYGELDF of SEQ ID NO: 59;

ii) the second antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO 92, wherein the VH domain comprises: (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, wherein X is D or E; and (c) CDR-H3 comprising the amino acid sequence GVGFFXH of SEQ ID NO. 78, wherein 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 to the amino acid sequence of SEQ ID NO:93, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence KAX of SEQ ID NO:79 ¹ EDIYNRX ² A, wherein X ¹ Is F or T and X ² Is R or L; (e) CDR-L2 comprising the amino acid sequence GATSLEH of SEQ ID NO. 80; and (f) CDR-L3 comprising SEQ ID NOID NO 81, wherein X is W or R;

or

H) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID No. 93, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence KAX of SEQ ID NO:79 ¹ EDIYNRX ² A, wherein X ¹ Is F or T and X ² Is R or L; (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, wherein X is W or R;

or

I) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO. 90, wherein the VH domain comprises: (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, wherein X is D or E; and (c) CDR-H3 comprising the amino acid sequence GVGFFXH of SEQ ID NO:78 wherein X is D or E;

and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% amino acid sequence of SEQ ID NO:9398%, 99% or 100% sequence identity, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence KAX of SEQ ID NO:79 ¹ EDIYNRX ² A, wherein X ¹ Is F or T and X ² Is R or L; (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, wherein X is W or R;

or

J) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO 92, wherein the VH domain comprises: (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, wherein X is D or E; and (c) CDR-H3 comprising the amino acid sequence GVGFFXH of SEQ ID NO. 78, wherein X is D or E;

or

K) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

L) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

M) i) said first antigen binding site comprises

ii) the second antigen binding site comprises

or

N) i) the first antigen binding site comprises

ii) the second antigen binding site comprises

or

O) i) said first antigen binding site comprises

ii) the second antigen binding site comprises

A VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO 91, wherein the VH domain comprises: (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, wherein X is D or E; and (c) CDR-H3 comprising the amino acid sequence GVGFFXH of SEQ ID NO:78 wherein X is D or E;

or

P) i) said first antigen binding site comprises

ii) the second antigen binding site comprises

and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:93, wherein the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence KAX of SEQ ID NO:79 ¹ EDIYNRX ² A, wherein X ¹ Is F or T and X ² Is R or L; (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, wherein X is W or R.

12. The bispecific antibody of any one of claims 8 to 11, wherein the bispecific antibody comprises an Fc domain of human IgG isotype, preferably of human IgG1 isotype.

13. The bispecific antibody according to any one of claims 8 to 11, wherein said bispecific antibody comprises a constant domain of human IgG isotype, preferably of human IgG1 isotype.

14. The bispecific antibody of any one of the preceding claims, wherein the bispecific antibody

i) Blockade of CCL2 binding to its receptor CCR2 in vitro (reporter gene assay, IC ₅₀ 0.5 nM); and/or

ii) inhibition of CCL 2-mediated chemotaxis (IC) of bone marrow cells in vitro ₅₀ 1.5 nM); and/or

iii) cross-react with cynomolgus monkey CCL2 and human CCL 2.

15. The bispecific antibody of any one of the preceding claims, wherein the bispecific antibody is non-cross-reactive with other CCL homologs.

16. The bispecific antibody of any one of the preceding claims, wherein the bispecific antibody binds human CCL2 at pH 7.4 with 10-fold greater affinity than at pH 5.8.

17. The bispecific antibody of any one of the preceding claims, wherein the bispecific antibody comprises a human IgG1 heavy chain constant domain comprising one or more of 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 a 330K; and/or

iii) M428L, N434A and/or Y436T; and/or

iv) Q438R and/or S440E.

18. An (isolated) antibody that binds (specifically) to human CCL2,

wherein the antibody comprises

A) A VH domain comprising: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO 57, wherein X is I or T; (b) CDR-H2 comprising the amino acid sequence GX of SEQ ID NO. 58 ¹ IX ² IFX ³ TANYAQKFQG, wherein X ¹ Is V, I or H, X ² Is P or H, and X ³ Is H or G;and (c) CDR-H3 comprising amino acid sequence YDAHYGELDF of SEQ ID NO: 59;

and

or

And

19. An (isolated) antibody that binds (specifically) to human CCL2,

wherein the antibody comprises

A) A VH domain comprising the amino acid sequence of SEQ ID NO 71;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75;

or

B) 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;

or

C) A VH domain comprising the amino acid sequence of SEQ ID NO 73;

and a VL domain comprising the amino acid sequence of SEQ ID NO 75;

or

D) 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;

or

E) 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

F) 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

G) 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

H) 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 94;

20. an isolated nucleic acid encoding the antibody of any one of the preceding claims.

21. A host cell comprising the nucleic acid of claim 20.

22. A method of producing an antibody comprising culturing the host cell of claim 21, thereby producing the antibody.

23. The method of claim 23, further comprising recovering the antibody from the host cell.

24. A pharmaceutical formulation comprising the bispecific antibody of any one of claims 1 to 17 and a pharmaceutically acceptable carrier.

25. The bispecific antibody according to any one of claims 1 to 17 for use as a medicament.

26. The bispecific antibody of any one of claims 1 to 17 for use in the treatment of cancer.

27. The bispecific antibody of any one of claims 1 to 17 for use in the treatment of an inflammatory or autoimmune disease.

28. Use of a bispecific antibody according to any one of claims 1 to 17 in the manufacture of a medicament.

29. The use of claim 28, wherein the medicament is for the treatment of cancer.

30. The use according to claim 28, wherein the medicament is for the treatment of an inflammatory disease or an autoimmune disease.

31. A method of treating an individual having cancer, the method comprising administering to the individual an effective amount of the bispecific antibody according to any one of embodiments 1 to 17.

32. A method of treating an individual having an inflammatory disease or an autoimmune disease, the method comprising administering to the individual an effective amount of the bispecific antibody according to any one of embodiments 1 to 17.