CN110483640B

CN110483640B - Humanized monoclonal antibody of interleukin-6R, and coding gene and application thereof

Info

Publication number: CN110483640B
Application number: CN201910644511.XA
Authority: CN
Inventors: 刘鸿君; 李耀东; 李娴
Original assignee: Beijing Huizhi Heyuan Biotechnology Co ltd
Current assignee: Huizhi Heyuan Biotechnology Suzhou Co ltd
Priority date: 2019-07-16
Filing date: 2019-07-16
Publication date: 2020-05-01
Anticipated expiration: 2039-07-16
Also published as: CN110483640A

Abstract

The present invention relates to an antibody against interleukin-6 receptor (IL-6R), a pharmaceutical composition or kit comprising the same, and uses thereof.

Description

Humanized monoclonal antibody of interleukin-6R, and coding gene and application thereof

Technical Field

The invention relates to the technical field of antibodies, in particular to a humanized monoclonal antibody of interleukin-6R, and a coding gene and application thereof.

Background

Interleukin-6 (IL-6) (also known as interferon- β 2, B cell differentiation factor, B cell stimulatory factor-2, hepatocyte stimulatory factor, hybridoma growth factor) is a multifunctional cytokine produced by a variety of different cell types that is involved in a number of biological processes including modulation of acute inflammatory responses, modulation of specific immune responses (including B cell and T cell differentiation), bone metabolism, thrombopoiesis, epidermal cell proliferation, menstruation, neuronal cell differentiation, neuroprotection, Aging, cancer and the inflammatory response seen in Alzheimer's disease, see A.Pasassotiotropoulos et al (2001), Neuroiogloyof Aging, 22: 863-one 871.

The biological function of IL-6 is mediated by the IL-6 receptor (IL-6R), which is widely expressed on the surface of a variety of cells. IL-6 can activate a series of signal protein molecules in cells through a receptor thereof, so as to realize the induction expression of IL-6 response genes. See e.sanz et al (2008), gia, 56: 190-199.

The cDNA for the human IL-6 receptor (hIL-6R) has a total length of about 5.0kb, a coding region of 1.4kb, and encodes a transmembrane glycoprotein of 468 amino acids, which contains a hydrophobic signal peptide of 19 amino acid residues. See Yamasakik, et al (1988), Science, 241: 825-828. IL-6R belongs to a member of the Class I cytokine receptor family, is a member of the immunoglobulin (Ig) superfamily, has high homology with other cytokine receptors, and has high interspecies homology.

The IL-6 receptor system consists of two distinct transmembrane glycoprotein chains, a high affinity specific ligand binding chain IL-6R α (gp80) and a non-ligand binding chain glycoprotein 130(gp130, IL-6R β subunit) as signal transducer, the expression of IL-6R α is limited to certain specific tissue cells, such as hepatocytes, neutrophils, macrophages and certain lymphocytes, whereas gp130 is expressed on almost all cell surfaces. IL-6R α is a molecule with a relative molecular weight of 80kDa, which binds directly to IL-6 to form a high affinity complex with gp130 after formation of the IL-6/IL-6R α complex. IL-6R α cytoplasmic domain is short, has no tyrosine kinase activity and no IL-6 signal transduction function, it requires the additional protein factor gp130 as a signal transducer in the IL-6 system, when extracellular IL-6 and IL-6R α interact to cause the gp130/IL-6R 7878 to be bound to the protein, but the protein is unable to form a signal transducer in the Rottt 7J 4578. the protein alone, see also the publication for the signal transduction of the protein of the type of IL-6.

Furthermore, there are soluble forms of IL-6R (i.e., sIL-6R) distributed in body fluids, restricted hydrolysis of sIL-6R, which may result from altered splicing of mRNA, IL-6R, which leads to shedding of IL-6R from the membrane, apoptotic neutrophils are one of the major sources of sIL-6R, see Mono-Julian, FA, et al (2001), Cell Mol Biol, 47: 583-597, which are involved in the development of IL-6 cells, and the mechanisms of IL-6 interaction with receptors, in addition to the above-mentioned pathways, there are also additional pathways through sIL-6R transduction, i.e., sIL-6R α binding to IL-6 to form a complex, which then binds to surface gpl30 cells, regulating intracellular signaling, which expands the range of IL-6 action (e.g., IL-6R α, synovial cells, etc., but not only through IL-6R α expression but also through IL-6R-6-7, which also through IL-7-IL-7, which are involved in the endothelial cells, NO, CD-7, which are induced by endothelial cells, CD-7, CD-NO, CD-7, which are induced by endothelial cells, which express, and induce apoptosis, which are induced by endothelial cells, and promote the endothelial Cell proliferation of IL-2-IL-11, and promote the endothelial Cell proliferation, mature, and promote the endothelial Cell-IL-11-IL-11, and promote the endothelial Cell-IL-11-IL-11-IL-11, and the endothelial Cell line, or macrophage-11, and the endothelial Cell line of endothelial Cell line, and the endothelial Cell line, which are induced by the endothelial Cell line of endothelial Cell line, or macrophage-11, and the endothelial Cell line of endothelial Cell line, the endothelial Cell line of endothelial Cell line, which are induced by the endothelial Cell line, the endothelial Cell line of endothelial Cell line, the endothelial Cell-11, the endothelial Cell-IL-macrophage-7, the endothelial Cell line, the endothelial Cell-macrophage-epithelial Cell line, the endothelial Cell line of the endothelial Cell line, the endothelial Cell line of the endothelial Cell line, the endothelial Cell line of the endothelial Cell line, the endothelial Cell-11, the endothelial Cell line of the endothelial Cell line, the endothelial Cell line of the endothelial Cell, the endothelial Cell line of the endothelial Cell, the endothelial Cell line of the endothelial.

The IL-6/IL-6R system has various biological activities, the expression and regulation of IL-6/IL-6R are related to the occurrence and development of a plurality of tumors, and the IL-6 level is proved to be increased in diseases related to IL-6, such as multiple myeloma, leukemia, kidney cancer, prostatic cancer, lymphoma, lung cancer, liver cancer, breast cancer, esophageal cancer and other tumors, and the expression abnormality or the affinity change of the IL-6R is also accompanied. In addition, the research also finds that the serum IL-6 level of the leukemia patient is far higher than that of the control group, and the remission stage is obviously reduced, which indicates that the IL-6 can be used as an index for judging the prognosis and recurrence of the tumor disease. IL-6/IL-6R are coordinated and act together on the body, and the abnormality of IL-6/IL-6R affects the stability of the whole internal environment, thereby causing the dysfunction of immune, hematopoietic and endocrine systems. See Lobo catti L, et al (2005), Clin Exp Med, 5: 112-116 Chopra, et al, (2004), mjfai, 60: 45-49; songur, et al, (2004), Tumori, 90: 196-200; blay, et al, (1992), Cancer Research, 52: 3317-3322; nikiteas, et al, (2005), worldj. gastereol, 11: 1639-1643; heikkila, et al, (2008), Eur J Cancer, 44: 937-945.

IL-6/IL-6R is thought to play a role in the development of a number of diseases and disorders, including but not limited to fatigue, cachexia, inflammatory diseases, autoimmune diseases, skeletal system diseases, fever, cancer, heart disease, obesity, diabetes, asthma, Alzheimer's disease, multicenter Castleman's disease, multiple sclerosis and rheumatoid arthritis. See, for example, WO2011/066374, WO2011/066371, WO2011/066378 and WO 2011/066369. To date, no monoclonal antibody against the IL-6R target has been marketed, except that Tocilizumab and Salulumab/Redox Kevzara (sarilumab) IL-6R monoclonal antibody drugs of Roche were approved in succession for marketing.

Disclosure of Invention

The present invention relates to the following aspects:

1. an antibody or antigen-binding fragment thereof, in particular, which binds IL-6R, preferably human IL-6R, wherein the antibody comprises:

HCDR1 comprising SEQ ID NO: 6, a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, or consisting thereof,

HCDR2 comprising SEQ ID NO:7, a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, and

HCDR3 comprising SEQ ID NO:8, a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, or consisting thereof,

and the antibody further comprises:

LCDR1 comprising SEQ ID NO: 9, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, or consisting thereof,

LCDR2 comprising SEQ ID NO:10, a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, and

LCDR3 comprising SEQ ID NO:11, a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, or consisting thereof.

In one embodiment, the antibody comprises:

(1) (i) a heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO:4, or

And SEQ ID NO:4 has at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or

And SEQ ID NO:4 with one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in (4), and

(ii) a light chain variable region comprising or consisting of the sequence:

SEQ ID NO:5, or

And SEQ ID NO:5, or a sequence having at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence depicted in seq id No. 5, or

And SEQ ID NO:5 with one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in (a);

(2) (i) a heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO:26, or

And SEQ ID NO:26 has at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or

And SEQ ID NO:26 with one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence shown in (a), and

(ii) a light chain variable region comprising or consisting of the sequence:

SEQ ID NO:27, or

And SEQ ID NO:27, or a sequence having at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence depicted in seq id No. 27, or

And SEQ ID NO:27 with one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutation(s) (preferably substitution(s), insertion(s) or deletion (s));

(3) (i) a heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO:26, or

(ii) a light chain variable region comprising or consisting of the sequence:

SEQ ID NO:41, or

And SEQ ID NO:41 has at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or

And SEQ ID NO:41 with one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); or (4) (i) a heavy chain variable region comprising or consisting of:

SEQ ID NO:26, or

(ii) a light chain variable region comprising or consisting of the sequence:

SEQ ID NO:49, or

And SEQ rD NO:49 has at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or

And SEQ ID NO:49 compared to an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions).

In one embodiment, the heavy chain variable region and the light chain variable region are each encoded by the following nucleotide sequences:

(1) (i) SEQ ID NO:22, or

And SEQ ID NO:22 has at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or

And SEQ ID NO:22 has one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative mutations (preferably substitutions, insertions or deletions) compared to the nucleotide sequence set forth in seq id no, and

(ii) SEQ ID NO:23, or

And SEQ ID NO:23, has at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or

And SEQ ID NO:23 with one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative mutations (preferably substitutions, insertions or deletions) compared to the nucleotide sequence set forth in seq id no;

(2) (i) SEQ ID NO:38, or

And SEQ ID NO:38 has at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or

And SEQ ID NO:38 with one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative mutations (preferably substitutions, insertions or deletions), and

(ii) SEQ ID NO:39, or

And SEQ ID NO:39, has at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or

And SEQ ID NO:39 with one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative mutations (preferably substitutions, insertions or deletions) compared to the nucleotide sequence set forth in (a);

(3) (i) SEQ ID NO:38, or

(ii) SEQ ID NO:47, or

And SEQ ID NO:47, or a sequence having at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence depicted in seq id no, or

And SEQ ID NO:47 with one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative mutations (preferably substitutions, insertions or deletions) compared to the nucleotide sequence set forth in seq id no; or

(4) (i) SEQ ID NO:38, or

(ii) SEQ ID NO:55, or

And SEQ ID NO:55, has at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or

And SEQ ID NO:55 with one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative mutations (preferably substitutions, insertions or deletions) compared to the nucleotide sequence set forth in seq id no.

In one embodiment, the antibody further comprises the framework regions FR-H1, FR-H2, FR-H3 and FR-H4 of the heavy chain variable region and the framework regions FR-L1, FR-L2, FR-L3 and FR-L4 of the light chain variable region, wherein

(1) FR-H1 comprises the amino acid sequence of SEQ ID NO:12, or an amino acid sequence identical to SEQ ID NO:12, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:12, or consists of an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in seq id no;

FR-H2 comprises the amino acid sequence of SEQ ID NO:13 or an amino acid sequence identical to SEQ ID NO:13, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:13, or consists of an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in (a);

FR-H3 comprises the amino acid sequence of SEQ ID NO:14 or an amino acid sequence corresponding to SEQ ID NO:14, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:14, or consists of an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in (a);

FR-H4 comprises the amino acid sequence of SEQ ID NO:15, or an amino acid sequence identical to SEQ ID NO:15, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:15 or consists of an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in (b); and

FR-L1 comprises the amino acid sequence of SEQ ID NO:16 or an amino acid sequence corresponding to SEQ ID NO:16, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:16, or consists of an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in (b); FR-L2 comprises the amino acid sequence of SEQ ID NO:17 or an amino acid sequence substantially identical to SEQ ID NO:17, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:17, or consists of an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in (b); FR-L3 comprises the amino acid sequence of SEQ ID NO:18 or an amino acid sequence corresponding to SEQ ID NO:18, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:18, or consists of an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in (a); FR-L4 comprises the amino acid sequence of SEQ ID NO:19 or an amino acid sequence substantially identical to SEQ ID NO:19, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:19, or consists of an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions);

(2) FR-H1 comprises the amino acid sequence of SEQ ID NO:28 or an amino acid sequence identical to SEQ ID NO:28, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:28, or consists of, an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in seq id no;

FR-H2 comprises the amino acid sequence of SEQ ID NO:29 or an amino acid sequence substantially identical to SEQ ID NO:29, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:29 compared to an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions);

FR-H3 comprises the amino acid sequence of SEQ ID NO:30 or an amino acid sequence corresponding to SEQ ID NO:30, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:30, or consists of an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in (b);

FR-H4 comprises the amino acid sequence of SEQ ID NO:31 or an amino acid sequence substantially identical to SEQ ID NO:31, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:31, or consists of an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in (b); and

FR-L1 comprises the amino acid sequence of SEQ ID NO:32 or an amino acid sequence substantially identical to SEQ ID NO:32, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:32, or consists of, an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in (b);

FR-L2 comprises the amino acid sequence of SEQ ID NO:33 or an amino acid sequence identical to SEQ ID NO:33, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:33, or consists of an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in seq id no;

FR-L3 comprises the amino acid sequence of SEQ ID NO:34 or an amino acid sequence corresponding to SEQ ID NO:34, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:30, or consists of an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in (b);

FR-L4 comprises the amino acid sequence of SEQ ID NO:34 or an amino acid sequence corresponding to SEQ ID NO:34, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:34, or consists of, one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions);

(3) FR-H1 comprises the amino acid sequence of SEQ ID NO:28 or an amino acid sequence identical to SEQ ID NO:28, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:28, or consists of, an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in seq id no;

FR-L1 comprises the amino acid sequence of SEQ ID NO:42, or an amino acid sequence identical to SEQ ID NO:42, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:42 compared to an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions);

FR-L2 comprises the amino acid sequence of SEQ ID NO:43, or an amino acid sequence substantially identical to SEQ ID NO:43, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:43, or consists of an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in (b);

FR-L3 comprises the amino acid sequence of SEQ ID NO:44, or an amino acid sequence identical to SEQ ID NO:44, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:44 compared to an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions);

FR-L4 comprises the amino acid sequence of SEQ ID NO:45, or an amino acid sequence substantially identical to SEQ ID NO:45, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:45, or consists of an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); or

(4) FR-H1 comprises the amino acid sequence of SEQ ID NO:28, or an amino acid sequence identical to SEQ ID NO:28, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:28, or consists of an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in seq id no;

FR-H2 comprises the amino acid sequence of SEQ ID NO:29, or an amino acid sequence identical to SEQ ID NO:29, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:29 compared to an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions);

FR-H3 comprises the amino acid sequence of SEQ ID NO:30, or an amino acid sequence identical to SEQ ID NO:30, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:30 or consists of an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in (b);

FR-H4 comprises the amino acid sequence of SEQ ID NO:31, or an amino acid sequence that is identical to SEQ ID NO:31, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:31 or consists of an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence shown in (b), and

FR-L1 comprises the amino acid sequence of SEQ ID NO:50, or an amino acid sequence substantially identical to SEQ ID NO:50, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:50 with or consisting of one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions);

FR-L2 comprises the amino acid sequence of SEQ ID NO:51, or an amino acid sequence identical to SEQ ID NO:51, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:51 compared to an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions);

FR-L3 comprises the amino acid sequence of SEQ ID NO:52, or an amino acid sequence identical to SEQ ID NO:52, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:52 with one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions);

FR-L4 comprises the amino acid sequence of SEQ ID NO:53, or an amino acid sequence identical to SEQ ID NO:53, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:53, or consists of an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions).

In one embodiment, the antibody comprises or consists of an amino acid sequence selected from the group consisting of the following heavy and light chain combinations:

(1) SEQ ID NO:2 and the amino acid sequence of SEQ ID NO: 3;

(2) SE ID NO:24 and the amino acid sequence of SEQ ID NO: 25;

(3) SEQ ID NO:24 and the amino acid sequence of SEQ ID NO: 40; or

(4) SEQ ID NO:24 and the amino acid sequence of SEQ ID NO: 48.

In one embodiment, the heavy and light chains are each encoded by the nucleotide sequences:

(1) SEQ ID NO:20 and the nucleotide sequence of SEQ ID NO: 21;

(2) SE ID NO:36 and the nucleotide sequence of SEQ ID NO: 37;

(3) SEQ ID NO:36 and the nucleotide sequence of SEQ ID NO: 46; or

(4) SEQ ID NO:36 and the nucleotide sequence of SEQ ID NO:54, or a nucleotide sequence of seq id no.

In one embodiment, the antibody is a humanized antibody, a chimeric antibody, or a multispecific antibody (e.g., bispecific antibody).

In one embodiment, the constant region of the antibody is humanized, preferably from human IgG, more preferably IgGl or IgG 4.

In one embodiment, the heavy chain constant region of the antibody is an Ig gamma-1 or Ig gamma-4 chain C region, preferably an Ig gamma-1 chain C region; the light chain constant region is the Ig kappa chain C region, more preferably GenBank ACCESSION No. access: ig kappa chain C region of P01834.

In one embodiment, the antigen isThe binding fragment is selected from the group consisting of Fab, Fab ', F (ab')₂Fd, Fv, dAb, Fab/c, Complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), diabodies, or domain antibodies.

In one aspect, the invention relates to an isolated polypeptide selected from the group consisting of:

(1) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 6, 7 and 8, wherein the polypeptide specifically binds to human IL-6R as part of an anti-human IL-6R antibody further comprising the amino acid sequence set forth in SEQ ID NO: 9, 10 and 11;

(2) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 9, 10 and 11, wherein the polypeptide specifically binds human IL-6R as part of an antibody against human IL-6R, said antibody further comprising the amino acid sequence set forth in SEQ ID NO: 6, 7 and 8;

(3) an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide specifically binds to human IL-6R as part of an anti-human IL-6R antibody further comprising a heavy chain variable region selected from the group consisting of SEQ ID NOs: 5 or a sequence having at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

(4) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:5 or a sequence having at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide specifically binds to human IL-6R as part of an anti-human IL-6R antibody further comprising an amino acid sequence selected from the group consisting of seq id NO:4 or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

(5) an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26 or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide specifically binds to human IL-6R as part of an anti-human IL-6R antibody further comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 or a sequence having at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

(6) an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 or a sequence having at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide specifically binds to human IL-6R as part of an anti-human IL-6R antibody further comprising an amino acid sequence selected from the group consisting of SEQ ID NO:26 or a sequence having at least 90%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

(7) an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26 or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide specifically binds to human IL-6R as part of an anti-human IL-6R antibody further comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41 or a sequence having at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

(8) an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41 or a sequence having at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide specifically binds to human IL-6R as part of an anti-human IL-6R antibody further comprising an amino acid sequence selected from the group consisting of SEQ ID NO:26 or a sequence having at least 90%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

(9) an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26 or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide specifically binds to human IL-6R as part of an anti-human IL-6R antibody further comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 49 or a sequence having at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence; or

(10) An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 49 or a sequence having at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide specifically binds to human IL-6R as part of an anti-human IL-6R antibody further comprising an amino acid sequence selected from the group consisting of SEQ ID NO:26 or a sequence having at least 90%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

(11) an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide specifically binds to human IL-6R as part of an anti-human IL-6R antibody further comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 or a sequence having at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

(12) an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 or a sequence having at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide specifically binds to human IL-6R as part of an anti-human IL-6R antibody further comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2 or a sequence having at least 90%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

(13) an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24 or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide specifically binds to human IL-6R as part of an anti-human IL-6R antibody further comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25 or a sequence having at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

(14) an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25 or a sequence having at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide specifically binds to human IL-6R as part of an anti-human IL-6R antibody further comprising an amino acid sequence selected from the group consisting of SEQ ID NO:24 or a sequence having at least 90%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

(15) an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24 or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide specifically binds to human IL-6R as part of an anti-human IL-6R antibody further comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 40 or a sequence having at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

(16) an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 40 or a sequence having at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide specifically binds to human IL-6R as part of an anti-human IL-6R antibody further comprising an amino acid sequence selected from the group consisting of SEQ ID NO:24 or a sequence having at least 90%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

(17) an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24 or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide specifically binds to human IL-6R as part of an anti-human IL-6R antibody further comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 or a sequence having at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence; or

(18) An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 or a sequence having at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide specifically binds to human IL-6R as part of an anti-human IL-6R antibody further comprising an amino acid sequence selected from the group consisting of SEQ ID NO:24 or a sequence having at least 90%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence.

In another aspect, the invention relates to an isolated polynucleotide encoding an isolated polypeptide of the invention.

In another aspect, the invention relates to a vector comprising an isolated polynucleotide of the invention.

In another aspect, the invention relates to a host cell comprising an isolated polynucleotide of the invention or a vector of the invention.

In another aspect, the invention relates to a method of making an antibody or antigen-binding fragment thereof of the invention comprising culturing a host cell of the invention.

In another aspect, the invention relates to an antibody conjugate comprising an antibody or antigen-binding fragment thereof according to the invention and a coupling moiety coupled thereto, preferably said coupling moiety is selected from the group consisting of a purification tag (e.g. a His-tag), a cytotoxic agent, a detectable label, a radioisotope, a chemiluminescent substance, a chromogenic substance, an enzyme or polyethylene glycol.

In another aspect, the invention relates to multispecific antibodies, preferably bispecific antibodies, comprising an antibody or antigen-binding fragment thereof according to the invention, and antibodies or antigen-binding fragments directed against other antigens and/or other antigenic epitopes.

In another aspect, the invention relates to a fusion protein comprising an antibody or antigen-binding fragment thereof described herein.

In another aspect, the present invention relates to a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the present invention, the antigen conjugate, the multispecific antibody or the fusion protein, optionally further comprising a pharmaceutically acceptable carrier and/or excipient.

In one embodiment, the pharmaceutical composition is in a dosage form suitable for administration by injection, for example, a dosage form suitable for intravenous injection.

In another aspect, the invention relates to a kit comprising the antibody or antigen-binding fragment thereof, the antigen conjugate, the multispecific antibody or the fusion protein of the invention, preferably the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof, the antigen conjugate, the multispecific antibody or the fusion protein of the invention; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a chemiluminescent substance, a chromogenic substance, an enzyme.

In another aspect, the invention relates to the antibody or antigen binding fragment thereof, the antigen conjugate, the multispecific antibody or the fusion protein in the preparation of a kit for detecting the presence or level of human IL-6R in a sample.

In another aspect, the invention relates to the use of said antibody or antigen binding fragment thereof, said antigen conjugate, said multispecific antibody or said fusion protein in the manufacture of a medicament for:

drugs that block the binding of human IL-6 to human IL-6R,

(ii) an agent that blocks the activity or down-regulates the level of human IL-6R,

drugs which inhibit gp130 signaling and decrease the phosphorylation level of its downstream signaling protein p-Stat3(Tyr705), or

Blocking the cytological response mediated by the binding of human IL-6 to IL-6R.

In another aspect, the invention relates to the use of said antibody or antigen-binding fragment thereof, said antigen conjugate, said multispecific antibody or said fusion protein for the preparation of a medicament for the prevention and/or treatment and/or co-treatment and/or diagnosis of fatigue, cachexia, inflammatory diseases, autoimmune diseases, skeletal system diseases, fever, cancer, heart disease, obesity, diabetes, asthma, alzheimer's disease, multicentric Castleman's disease, multiple sclerosis and rheumatoid arthritis.

In another aspect, the invention relates to said antibody or antigen binding fragment thereof, said antigen conjugate, said multispecific antibody or said fusion protein for use in the prevention and/or treatment and/or adjuvant therapy and/or diagnosis of a disease associated with IL-6R, such as obesity, an immune-related disease, a cardiovascular disease, an infectious disease, a malignant disease, a neurological disease, a wound, a trauma or tissue damage or an associated chronic disorder,

wherein it is preferred that, among others,

the IL-6R related immune related diseases include but are not limited to at least one

(1) Respiratory diseases

Obstructive airway disease; asthma; bronchitis; acute, allergic, atrophic rhinitis and chronic rhinitis; membranous rhinitis; seasonal rhinitis; sarcoidosis, farmer's lung and related diseases, adult respiratory distress syndrome, allergic pneumonia, fibrotic lung and idiopathic interstitial pneumonia; chronic lung disease of newborn;

(2) bones and joints

Rheumatoid arthritis, childhood rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, juvenile chronic arthritis, seronegative spondyloarthritis (including psoriatic arthritis, ankylosing spondylitis and reiter's disease), behcet's disease, sjogren's syndrome, systemic sclerosis, osteoarthritis, gout, osteolysis;

(3) skin(s)

Psoriasis, allergic contact dermatitis, atopic dermatitis, other eczematous dermatoses, seborrheic dermatitis, lichen planus, scleroderma, pemphigus, bullous pemphigoid, epidermolysis bullosa, urticaria, rubella, xeroderma (angiodermas), vasculitis, erythema, hypereosinophilia of the skin, uveitis, alopecia areata, allergic conjunctivitis, and vernal vemal conjuctivitis;

(4) gastrointestinal tract

Gastric ulcer, inflammatory bowel disease, ulcerative colitis, coeliac disease, proctitis, eosinophilic gastroenteritis, mastocytosis, crohn's disease, ulcerative colitis, antiphospholipid syndrome, food-related allergies that produce effects remote from the viscera, such as migraine, rhinitis and eczema;

(5) graft rejection, graft versus host disease, allograft rejection of any organ or tissue (kidney, heart, liver, pancreas, lung, bone marrow, skin, cartilage, bone, small intestine, fetal thymus, parathyroid, cornea), xenograft rejection of any organ or tissue

(6) Other tissue and systemic diseases

Cachexia, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, antiphospholipid syndrome, iridocyclitis/uveitis/optic neuritis, systemic vasculitis/wegener's granulomatosis, sarcoidosis, orchitis/vasectomy reversal process, allergic/atopic diseases, allergic contact dermatitis, systemic inflammatory response syndrome, septicemia syndrome, gram positive sepsis, gram negative sepsis, culture negative sepsis, fungal sepsis, neutropenia fever, urinary sepsis, meningococcemia, trauma/hemorrhage, burns, ionizing radiation exposure, acute pancreatitis, alcohol-induced hepatitis, chronic inflammatory pathology, sterile relaxation of surgical implants, sarcoidosis, sickle cell anemia, diabetes, nephropathy, atopic diseases, hypersensitivity reactions, hay fever, endometriosis, pernicious anemia, hemolytic diseases, thrombocytopenia (thrombocytopenia), anti-receptor hypersensitivity reactions, graves ' disease, raynaud's disease (B-type insulin, atheroma, acute lymphoblastic encephalopathy, hypothyroidism, lymphoblastic encephalopathy, endocrine leukopenia, nephrosis, lymphoblastic disease, endocrine-polycythemia, endocrine-myelodysplasia, endocrine-macrophage-and other diseases, endocrine dyscrasia, endocrine-macrophage diseases, endocrine-macrophage diseases, and other diseases, such as, endocrine diseases, and endocrine diseases, such as acute lymphomatosis, and endocrine diseases, and endocrine dyscraving diseases;

the cardiovascular disease includes, but is not limited to, at least one cardiac stun syndrome (cardiostun syndrome), myocardial infarction, congestive heart failure, stroke, ischemic attack, hemorrhage, acute coronary syndrome, arteriosclerosis, atherosclerosis, restenosis, diabetes, diabetic macular edema, diabetic atherosclerotic disease, hypertension, arterial hypertension, renovascular hypertension, syncope, shock, cardiovascular syphilis, heart failure, pulmonary (idiopathic) heart disease, primary pulmonary hypertension, arrhythmia, ectopic pulsatility, atrial flutter, atrial fibrillation (persistent or paroxysmal), post-perfusion syndrome, cardiopulmonary bypass inflammatory response, turbulent or multi-atrial tachycardia, regular narrow s qrtachycardia, specific arrhythmia, ventricular fibrillation, bundle of cardiac arrhythmias (hisbundlearhrhmias), Atrioventricular block, bundle branch block, ischemic conditions of the myocardium, coronary artery disease, angina pectoris, myocardial infarction, cardiomyopathy, dilated congestive cardiomyopathy, restrictive cardiomyopathy, valvular heart disease, endocarditis, pericardial disease, cardiac tumors, aortic and peripheral aneurysms, aortic dissection, inflammation of the aorta, occlusion of the abdominal aorta and its branches, peripheral vascular conditions, occlusive arterial disorders, peripheral atherosclerotic diseases, thromboangiitis obliterans, functional peripheral arterial disorders, raynaud's phenomenon and disease, cyanosis of hands and feet, erythromelalgia, venous diseases, thrombophlebitis, varicose veins, arteriovenous fistulas, lymphedema, lipoedema, unstable angina, reperfusion injury, post-pump syndrome, ischemia-reperfusion injury;

the IL-6R-associated infectious disease includes, but is not limited to, at least one of: acute or chronic bacterial infections, acute or chronic parasitic or infectious processes, including bacterial, viral and fungal infections, HTV infections/HIV neuropathy, meningitis, hepatitis (e.g. type a, type b or type c, etc.), septic arthritis, peritonitis, pneumonia, epiglottitis, e.coli 0157: h7, hemolytic uremic syndrome/thrombolytical thrombocytopenic purpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy, toxic shock syndrome, streptococcal myositis, gas gangrene, mycobacterium tuberculosis, mycobacterium avium intracellular parasitic bacteria, pneumocystis carinii pneumonia, pelvic inflammatory disease, orchitis/epididymitis, legionella, lyme disease, influenza a, epstein barr virus, virus-related hemophagocytic syndrome, viral encephalitis/aseptic meningitis, enterovirus type 71 hand-foot-and-mouth disease;

said IL-6R-associated malignancies include but are not limited to at least one of: leukemia, Acute Lymphoblastic Leukemia (ALL), acute lymphocytic leukemia, B-cell, T-cell or FAB ALL, Acute Myelogenous Leukemia (AML), acute myelogenous leukemia, Chronic Myelogenous Leukemia (CML), Chronic Lymphocytic Leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal cancer, pancreatic cancer, nasopharyngeal cancer, malignant histiocytosis, extratumoral syndrome/malignant hypercalcemia (idiopathic) syndrome, solid tumors, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head cancer, neck cancer, hereditary non-polyposis, Hodgkin's lymphoma, Liver cancer, lung cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, testicular cancer, adenocarcinoma, sarcoma, malignant melanoma, hemangioma, tumor metastatic disease, cancer-related bone resorption, cancer-related bone pain, etc.; inhibition of cancer metastasis; improvement in cancer cachexia;

the IL-6 associated neurological disorders include, but are not limited to, at least one of: neurodegenerative diseases, multiple sclerosis, migraine, AIDS dementia complex, demyelinating diseases, e.g. multiple sclerosis and acute transverse myelitis; extrapyramidal and cerebellar disorders, such as corticospinal lesions; disorders of the basal ganglia; dyskinesias with hyperkinesias, such as huntington's chorea and senile chorea; drug-induced dyskinesias, such as those induced by drugs that block CNS dopamine receptors; hypokinetic movement disorders, such as parkinson's disease; progressive anterior nuclear Palsy (progressive supra nuclear Palsy); structural damage to the cerebellum; spinocerebellar degeneration, such as spinal ataxia, friedreich's ataxia, cerebellar cortical degeneration, multiple system degenerative diseases (Mencel, Dejerine-Thomas, Shi-Drager and Machado-Joseph); systemic disorders (refsum's disease, abetalipoprotemia, ataxia, telangiectasia and mitochondrial multisystem disorders); demyelinating core disorders (demyelinating core disorders), such as multiple sclerosis, acute transverse myelitis; and motor unit disorders such as neuronal muscular atrophy (anterior horn cell degeneration, such as amyotrophic lateral sclerosis, infantile spinal muscular atrophy, and juvenile spinal muscular atrophy); alzheimer's disease; down syndrome in middle aged; diffuse Lewy body disease; lewy-type senile dementia; Wernike-Korsakov syndrome; chronic alcohol intoxication; creutzfeldt-jakob disease; subacute sclerosing panencephalitis, Hallerrorden-Spatz disease; dementia pugilistica; neurotrauma (e.g. spinal cord injury, brain injury, concussion, repetitive concussion); pain; inflammatory pain; autism disorder; depression and major depressive disorder; stroke; cognitive disorders; epilepsy;

said IL-6R-associated trauma, trauma or tissue damage or associated chronic disorders include, but are not limited to, at least one of: physical injury or trauma associated with oral surgery including periodontal surgery, tooth extraction, endodontic treatment, insertion of dental implants, application of dental restorations, and use; or wherein the wound is selected from the group consisting of a sterile wound, a contusion wound, a cut wound, a laceration wound, a non-penetrating wound, an open wound, a penetrating wound, a puncture wound, an infected wound, an infarct, and a subcutaneous wound; or wherein the wound is selected from the group consisting of an ischemic ulcer, a fistula, a severe bite, a thermal burn, and a donor site wound; or wherein the wound is an aphthous wound, a traumatic wound or a herpes associated wound.

In another aspect, the invention relates to an in vivo or in vitro method comprising the step of administering to a subject in need thereof a cell comprising said antibody or antigen-binding fragment thereof, said antigen conjugate, said multispecific antibody or said fusion protein, or an effective amount of said antibody or antigen-binding fragment thereof, said antigen conjugate, said multispecific antibody or said fusion protein, said method selected from the group consisting of:

blocking the combination of human IL-6 and human IL-6R,

blocking human IL-6R activity or down-regulating the level thereof,

inhibiting gp130 signal transduction and reducing the phosphorylation level of downstream signaling protein p-Stat3(Tyr705), or

Blocking the cell biological reaction mediated by the combination of human IL-6 and IL-6R.

In another aspect, the invention relates to a method for the prevention and/or treatment and/or co-treatment and/or diagnosis of diseases associated with IL-6R, such as obesity, immune-related diseases, cardiovascular diseases, infectious diseases, malignant diseases, neurological diseases, trauma or tissue damage or associated chronic disorders, comprising administering to a subject in need thereof said antibody or antigen-binding fragment thereof, said antigen conjugate, said multispecific antibody or said fusion protein,

wherein it is preferred that, among others,

(1) Respiratory diseases

(2) bones and joints

(3) skin(s)

(4) gastrointestinal tract

(6) Other tissue and systemic diseases

the IL-6R associated neurological disorders include, but are not limited to, at least one of: neurodegenerative diseases, multiple sclerosis, migraine, AIDS dementia complex, demyelinating diseases, e.g. multiple sclerosis and acute transverse myelitis; extrapyramidal and cerebellar disorders, such as corticospinal lesions; disorders of the basal ganglia; dyskinesias with hyperkinesias, such as huntington's chorea and senile chorea; drug-induced dyskinesias, such as those induced by drugs that block CNS dopamine receptors; hypokinetic movement disorders, such as parkinson's disease; progressive anterior nuclear Palsy (progressive supra nuclear Palsy); structural damage to the cerebellum; spinocerebellar degeneration, such as spinal ataxia, friedreich's ataxia, cerebellar cortical degeneration, multiple system degenerative diseases (Mencel, deierin-Thomas, Shi-Drager and Machado-Joseph); systemic disorders (refsum's disease, abetalipoprotemia, ataxia, telangiectasia and mitochondrial multisystem disorders); demyelinating core disorders (demyelinating core disorders), such as multiple sclerosis, acute transverse myelitis; and motor unit disorders such as neuronal muscular atrophy (anterior horn cell degeneration, such as amyotrophic lateral sclerosis, infantile spinal muscular atrophy, and juvenile spinal muscular atrophy); alzheimer's disease; down syndrome in middle aged; diffuse Lewy body disease; lewy-type senile dementia; Wernike-Korsakov syndrome; chronic alcohol intoxication; creutzfeldt-jakob disease; subacute sclerosing panencephalitis, Hallerrorden-Spatz disease; dementia pugilistica; neurotrauma (e.g. spinal cord injury, brain injury, concussion, repetitive concussion); pain; inflammatory pain; autism disorder; depression and major depressive disorder; stroke; cognitive disorders; epilepsy;

said IL-6-associated trauma, trauma or tissue damage or associated chronic disorders include, but are not limited to, at least one of: physical injury or trauma associated with oral surgery including periodontal surgery, tooth extraction, endodontic treatment, insertion of dental implants, application of dental restorations, and use; or wherein the wound is selected from the group consisting of a sterile wound, a contusion wound, a cut wound, a laceration wound, a non-penetrating wound, an open wound, a penetrating wound, a puncture wound, an infected wound, an infarct, and a subcutaneous wound; or wherein the wound is selected from the group consisting of an ischemic ulcer, a fistula, a severe bite, a thermal burn, and a donor site wound; or wherein the wound is an aphthous wound, a traumatic wound or a herpes associated wound.

In particular, the invention relates to the following sequences:

SEQ ID NO:2 the amino acid sequence of heavy chain H of the murine monoclonal antibody;

SEQ ID NO:3 the amino acid sequence of the light chain L of the murine monoclonal antibody;

SEQ ID NO:4 the variable region amino acid sequence of heavy chain H of the murine monoclonal antibody;

SEQ ID NO:5 the variable region amino acid sequence of a light chain L of a murine monoclonal antibody;

SEQ ID NO: 6: CDR1 sequence of heavy chain H of murine monoclonal antibody;

SEQ ID NO: CDR2 sequence of heavy chain H of the murine monoclonal antibody;

SEQ ID NO: CDR3 sequence of heavy chain H of the 8 murine monoclonal antibody;

SEQ ID NO: CDR1 sequence of light chain L of the murine monoclonal antibody;

SEQ ID NO: the CDR2 sequence of light chain L of the 10 murine monoclonal antibody;

SEQ ID NO:11 CDR3 sequence of murine monoclonal antibody light chain L.

SEQ ID NO: FR1 sequence of heavy chain H of monoclonal antibody 12 of murine origin

SEQ ID NO: FR2 sequence of heavy chain H of monoclonal antibody derived from 13 mice

SEQ ID NO: FR3 sequence of heavy chain H of 14 murine monoclonal antibody

SEQ ID NO: FR4 sequence of heavy chain H of monoclonal antibody 15 murine

SEQ ID NO: FR1 sequence of light chain L of 16 murine monoclonal antibody

SEQ ID NO: FR2 sequence of light chain L of 17 murine monoclonal antibody

SEQ ID NO: FR3 sequence of 18 murine monoclonal antibody light chain L

SEQ ID NO: FR4 sequence of light chain L of 19 murine monoclonal antibody

SEQ ID NO: nucleotide sequence of heavy chain H of 20 murine monoclonal antibody

SEQ ID NO: nucleotide sequence of 21 murine monoclonal antibody light chain L

SEQ ID NO: nucleotide sequence of variable region of heavy chain H of 22 murine monoclonal antibody

SEQ ID NO: nucleotide sequence of variable region of 23 murine monoclonal antibody light chain L

SEQ ID NO: the amino acid sequence of the heavy chain H3 of the 24 humanized monoclonal antibody;

SEQ ID NO:25 the amino acid sequence of humanized monoclonal antibody light chain L1;

SEQ ID NO: the variable region amino acid sequence of the heavy chain H3 of the humanized monoclonal antibody of 26;

SEQ ID NO: the variable region amino acid sequence of light chain L1 of the humanized monoclonal antibody 27;

SEQ ID NO: FR1 sequence of 28 humanized monoclonal antibody heavy chain H3

SEQ ID NO: FR2 sequence of heavy chain H3 of 29 humanized monoclonal antibody

SEQ ID NO: FR3 sequence of heavy chain H3 of 30 humanized monoclonal antibody

SEQ ID NO: FR4 sequence of heavy chain H3 of 31 humanized monoclonal antibody

SEQ ID NO: FR1 sequence of light chain L1 of 32 humanized monoclonal antibody

SEQ ID NO: FR2 sequence of light chain L1 of 33 humanized monoclonal antibody

SEQ ID NO: FR3 sequence of light chain L1 of 34 humanized monoclonal antibody

SEQ ID NO: FR4 sequence of 35 humanized monoclonal antibody light chain L1

SEQ ID NO:36 humanized monoclonal antibody heavy chain H3 nucleotide sequence

SEQ ID NO: nucleotide sequence of light chain L1 of 37 humanized monoclonal antibody

SEQ ID NO:38 variable region nucleotide sequence of heavy chain H3 of humanized monoclonal antibody

SEQ ID NO:39 variable region nucleotide sequence of light chain L1 of humanized monoclonal antibody

SEQ ID NO:40 the amino acid sequence of humanized monoclonal antibody light chain L2;

SEQ ID NO:41 the variable region amino acid sequence of humanized monoclonal antibody light chain L2;

SEQ ID NO: FR1 sequence of light chain L2 of 42 humanized monoclonal antibody

SEQ ID NO: FR2 sequence of light chain L2 of 43 humanized monoclonal antibody

SEQ ID NO: FR3 sequence of light chain L2 of humanized monoclonal antibody 44

SEQ ID NO: FR4 sequence of light chain L2 of humanized monoclonal antibody 45

SEQ ID NO: nucleotide sequence of light chain L2 of 46 humanized monoclonal antibody

SEQ ID NO: nucleotide sequence of variable region of 47 humanized monoclonal antibody light chain L2

SEQ ID NO: amino acid sequence of 48 humanized monoclonal antibody light chain L3;

SEQ ID NO: the variable region amino acid sequence of light chain L3 of the humanized monoclonal antibody;

SEQ ID NO: FR1 sequence of 50 humanized monoclonal antibody light chain L3

SEQ ID NO: FR2 sequence of light chain L3 of 51 humanized monoclonal antibody

SEQ ID NO: FR3 sequence of light chain L3 of humanized monoclonal antibody 52

SEQ ID NO: FR4 sequence of 53 humanized monoclonal antibody light chain L3

SEQ ID NO: nucleotide sequence of light chain L3 of 54 humanized monoclonal antibody

SEQ ID NO: nucleotide sequence of variable region of light chain L2 of 55 humanized monoclonal antibody

IL-6/IL-6R is thought to play a role in the development of a number of diseases and disorders, including but not limited to fatigue, cachexia, inflammatory diseases, autoimmune diseases, skeletal system diseases, fever, cancer, heart disease, obesity, diabetes, asthma, Alzheimer's disease, multicenter Castleman's disease, multiple sclerosis and rheumatoid arthritis.

Unlike the marketed mechanism of action of Tocillizumab, the monoclonal antibody of the present invention acts by inhibiting the IL-6/IL-6R/gp130 ternary complex, whereas Tocillizumab acts by inhibiting the IL-6/IL-6R ternary complex.

Description of the drawings:

FIG. 1: western blot of hybridoma cell lines reducing the phosphorylation level of downstream signaling protein p-Stat3(Tyr 705).

FIG. 2: affinity of the humanized monoclonal antibody to human IL-6R.

FIG. 3: the humanized antibody inhibits the binding of IL-6 to IL-6R.

FIG. 4: the humanized monoclonal antibody inhibits the proliferation of DS-1 cells.

FIG. 5: humanized monoclonal antibodies HZ-0412a and b inhibit the phosphorylation western pattern of IL-6 stimulated p-Stat3(Tyr 705).

FIG. 6: the humanized monoclonal antibody HZ-0412c inhibits the phosphorylation western pattern of IL-6 stimulated p-Stat3(Tyr 705).

FIG. 7: phosphorylation ELISA of p-Stat3(Tyr705) stimulated by IL-6 was inhibited by humanized monoclonal antibodies.

FIG. 8: the humanized antibody inhibits SAA secretion from rhIL-6 stimulated HepG2 cells.

FIG. 9: the humanized antibody and various species of IL-6R cross reaction, wherein the left figure is humanized antibody to rhesus monkey IL-6R cross reaction; the right panel shows the cross-reaction of the humanized antibody to rat IL-6R

Detailed Description

The present invention is described in detail below by way of examples. It will be understood by those of ordinary skill in the art that the following examples are for illustrative purposes only. The spirit and scope of the present invention are defined by the appended claims.

EXAMPLE 1 preparation of human IL-6R

Human IL-6R eukaryotic expression vector is constructed, 293F cell is transfected, and culture supernatant is purified by a Protein A column to prepare human IL-6R Protein for mouse immunization, clone screening and function identification.

① construction of human IL-6R eukaryotic expression vector

The human IL-6R sequence starts from 1-Met of natural human IL-6R, 372 amino acids (SEQ ID NO: 1) are added to 372-Met, a mouse Fc fragment (mFc) is added at the C end (Accession number: AJ487681, Allle or Gene: IGHG1 × 01, SEQ ID NO: 56), and can be combined with recombinant protein A in a protein A column, so that purification can be carried out through affinity chromatography, and two enzyme cutting sites of HindIII and Not I are added at the two ends to obtain the rhIL-6R-mFc (the nucleotide sequence is SEQ ID NO: 57, and the amino acid sequence is SEQ ID NO: 58). Human rhIL-6R-mFc and an expression vector pcDNA3.1(+) (V790-20, Thermo) are subjected to double enzyme digestion through HindIII and Not I, a target fragment and an expression vector fragment of the human rhIL-6R-mFc are recovered, are connected and transformed, positive clone is identified through a PCR (polymerase chain reaction) and enzyme digestion method, and finally the correctness of the expression vector is verified through sequencing, so that the expression vector is named as pcDNA3.1(+) -rhIL-6R-mFc. Plasmids were extracted for transformation using a plasmid extraction kit.

② expression of rhIL-6R-mFc fusion gene in 293F cells

24 hours before transfection, 293F (Kjeldahl) was diluted to a density of 3, 0X 10 with 293 medium (Kjeldahl, K03252)⁶Individual cells/ml. At 130 rpm of constant temperature shaking table, 37 ℃ and 5% CO₂Culturing under conditions such that the cell density (by cell plate count) on the day of transfection is 4.0-6.0X 10⁶Individual cells/ml. To ensure optimal transfection efficiency, the cell viability (trypan blue staining) should be greater than 97%.

(taking 100ml of cell suspension transfection as an example), two 15ml sterile centrifuge tubes are prepared, 5ml of KPM (Kjeldahl, K03125L) and 100 mu g of sterile plasmid (pcDNA3.1(+) -rhIL-6R-mFc) DNA are added into one of the tubes, and the mixture is gently blown and beaten and mixed evenly; adding 5ml KPM and 500 μ l TA-293 (K20001) transfection reagent into the other branch, and gently blowing, beating and mixing; transferring all liquid in the centrifuge tube containing the transfection reagent into the centrifuge tube containing the plasmid, and gently blowing, beating and uniformly mixing; standing for 10 minutes at room temperature to prepare a plasmid-vector compound; taking out the cells from the constant temperature shaking table, adding the prepared plasmid-vector complex while shaking, and returning CO₂Culturing in a constant temperature shaking table. After 24 hours of transfection, 600 μ l 293 cell protein expression enhancer (KE-293) (Kjecery, K30001) and transient transfection nutrition additive (KT-Feed 50 ×) (Kjecery, K40001) can be added to increase the product expression; the supernatant was collected at about 5 days after transfection, centrifuged at 9000 rpm for 20 minutes by a refrigerated centrifuge, and the supernatant was collected for the next protein purification.

③ purification of rhIL-6R-mFc

The supernatant of the above antigen-containing 293F cells was centrifuged, captured using a protein a (protein a) column (GEHealthcare Bio-Sciences, 17-5080-02), eluted with 50mM citric acid-sodium citrate buffer (pH 3.0), the eluate (1.0 ml/tube) was collected, neutralized to neutrality by adding 50 μ l of 1M Tris-HCl buffer (pH 8.0), and dialyzed against phosphate-buffered saline (PBS) using a 10K dialysis membrane (general, M1915), and then the protein content was measured at 280 nm. Filtering, sterilizing, and storing at-80 deg.C.

Example 2 immunization of mice and determination of antibody titers in serum

Balb/c mice were immunized using rhIL-6R-mFc as antigen. The immunizing antigen (rhIL-6R-mFc) was obtained from example 1, Balb/c mice purchased from Tokyo Torrewa laboratory animal technology, Inc. The immunization route is subcutaneous multipoint injection, the immunization dose is 50 mug/200 mug/mouse, and 6 mice are immunized in total. The first immunization 50. mu.g of rhIL-6R-mFc were mixed with 100. mu.l of Freund's complete adjuvant (Sigma, F5881), the second and third immunization 50. mu.g of rhIL-6R-mFc were mixed with 100. mu.l of Freund's incomplete adjuvant (Sigma, F5506), and the fourth (booster) immunization was performed with 100. mu.g of rhIL-6R-mFc without adjuvant. The four immunization times were day 0, 14, 28, and 42, respectively.

After the third immunization (day 35), blood was collected from the eyeballs of 6 mice, and the titer of the anti-human IL-6R antibody in the serum of the immunized mice was measured by ELISA. First, the mixture was coated with a coating buffer (0.05M Na)₂CO₃-NaHCO₃pH 9.6) (CB) diluted rhIL-6R-hFc (expressed and synthesized by beijing huizhi and yurt biotechnology limited, nucleotide sequence of SEQ ID NO: 59, the amino acid sequence is SEQ ID NO: 60) to 1. mu.g/ml, 100. mu.l/well was added to a 96-well ELISA plate (Costar, 2592) at 4 degrees overnight. The next day, plates were washed 2 times with PBST (0.5 ‰), and blocked for 2 hours at room temperature by adding blocking solution (3% BSA in 1 × PBS). The plate was washed 1 time and the mouse serum was washed with PBS from 1: 1000 start 3 fold dilution, blank wells PBS and negative mouse serum, 100 μ L/well add to ELISA plate, incubate 1 hour at room temperature, wash plate 3 times, add to final concentration of 1 μ g/ml goat anti-mouse IgG (H + L) -HRP (proteinctech, SA00001-1), incubate 1 hour at room temperature. The plate was washed 4 times, and TMB (Zuman Bio, ZD311) color developing solution was added to develop color at room temperature for 10-20 minutes, and stop solution was added to read the absorbance at a wavelength of 450nm on a microplate reader (BioTek, ELx 808). Positive clones were defined as having an OD value greater than 2-fold that of the blank wells, and the higher the OD value at the highest dilution of the serum, indicating greater immunoreactivity to human IL-6R.

After the fourth immunization, the 3 rd mouse with the highest serum titer was selected for the subsequent preparation of hybridoma cells.

Example 3 preparation of hybridomas

After the last booster (day 42), spleen from mouse # 3 was taken, milled in saline and a B cell-rich suspension was taken and subjected to cell fusion with myeloma cell SP2/0 under the action of fusion agent PEG (Sigma, P7181). The fused cells were divided into 15 96-well cell culture plates in 20% fetuses containing HAT (Sigma, H0262)Bovine serum RPMI-1640 whole culture medium (Thermo, 31800089) in 5% CO₂And cultured at 37 ℃ for one week.

Example 4 screening of hybridoma Positive clones

① enzyme-linked immunosorbent assay (ELISA) screening of hybridoma positive clones with strong binding activity to antigen hIL-6R

Recombinant protein human rhIL-6R-hFc coated ELISA plate for the first round of positive cell strain screening. After the first round of screening, 80 positive hybridoma monoclonals with OD value > 1.0 were selected.

② Western immunoblotting (Western Blot) for screening hybridoma positive clones having strong neutralizing activity against rhIL-6R

Taking cell supernatants of different hybridoma positive clones, and adding DLD-1 cells respectively

CCL-221^TMAnd incubating at 37 ℃ for 2 hours, adding 10ng/ml rhIL-6-His (which is expressed and synthesized by Beijing Virginian and Yuan biotechnology Co., Ltd., and has a nucleotide sequence of SEQ ID NO: 61 and an amino acid sequence of SEQ ID NO: 62) into each well, incubating at 37 ℃ for 30 minutes, removing Cell supernatant, washing with PBS for 3 times, adding RIPA lysate to lyse the cells, collecting protein, performing SDS-PAGE electrophoresis on a protein sample, detecting the phosphorylation level of p-STAT3(Tyr705) (Cell Signaling, 52075) by Western Blot, and using β actin as a control (see FIG. 1).

As can be seen from FIG. 1, we screened a hybridoma positive clone with strong neutralizing activity, which can block IL-6 from binding to receptor IL-6R, inhibit gp130 signal transduction, and reduce the phosphorylation level of downstream signal protein p-Stat3(Tyr 705).

Example 5 obtaining of rhIL-6R murine monoclonal antibody

The total number of hybridoma cells having both binding and neutralizing activities in example 4 was cultured to 10⁷The cells were harvested by centrifugation at 1000rpm for 10 minutes and total RNA was extracted using Trizol kit (CWBBio, CW 0580S). First strand cDNA (CWBio, CW0744M) is synthesized using the RNA as a template, and hybridoma cells are amplified using the first strand cDNA as a subsequent templateThe corresponding variable region DNA sequence. The primer sequences used in the amplification reaction are complementary to the first framework and constant regions of the antibody variable region, reference (Larrick, J.W., et al (1990), Scand.J.Immunol., 32: 121-. Taq enzyme used (NEB, M0491S).

The sequence of the mouse monoclonal antibody secreted by the hybridoma positive clone is as follows:

SEQ ID NO: 6: CDR1 sequence of heavy chain H of murine monoclonal antibody;

SEQ ID NO: CDR2 sequence of heavy chain H of the murine monoclonal antibody;

SEQ ID NO: CDR3 sequence of heavy chain H of the 8 murine monoclonal antibody;

SEQ ID NO: CDR1 sequence of light chain L of the murine monoclonal antibody;

SEQ ID NO:11 CDR3 sequence of murine monoclonal antibody light chain L.

SEQ ID NO: FR3 sequence of heavy chain H of 14 murine monoclonal antibody

SEQ ID NO: FR4 sequence of heavy chain H of monoclonal antibody 15 murine

SEQ ID NO: FR1 sequence of light chain L of 16 murine monoclonal antibody

SEQ ID NO: FR2 sequence of light chain L of 17 murine monoclonal antibody

SEQ ID NO: FR3 sequence of 18 murine monoclonal antibody light chain L

SEQ ID NO: FR4 sequence of light chain L of 19 murine monoclonal antibody

SEQ ID NO: nucleotide sequence of 21 murine monoclonal antibody light chain L

Meanwhile, 6-8 weeks old Balb/c mice were intraperitoneally injected with 0.5ml Freund's incomplete adjuvant (Sigma, F5506) 7 days after injection, hybridoma cells having both binding and neutralizing activities of example 4 were injected, cultured to logarithmic phase, and adjusted to a cell concentration of 1X10 with PBS⁶～2X10⁶Individual cells/ml, i.p. (0.5 ml/mouse), hybridomas were grown in the abdominal cavity of mice and ascites fluid was produced. The abdominal cavity of the mouse can be enlarged 7-10 days after the hybridoma cells are injected, ascites of the mouse is collected, and the ascites is subjected to affinity purification by a proteinA A column to obtain the corresponding murine antibody.

Example 6 humanization and Performance validation of murine antibodies

According to the variable region sequence of the antibody secreted by the hybridoma cell, the humanized modification is carried out, and the specific sequence is obtained as follows:

SEQ ID NO: FR1 sequence of 28 humanized monoclonal antibody heavy chain H3

SEQ ID NO: FR2 sequence of heavy chain H3 of 29 humanized monoclonal antibody

SEQ ID NO: FR3 sequence of heavy chain H3 of 30 humanized monoclonal antibody

SEQ ID NO: FR4 sequence of heavy chain H3 of 31 humanized monoclonal antibody

SEQ ID NO: FR1 sequence of light chain L1 of 32 humanized monoclonal antibody

SEQ ID NO: FR2 sequence of light chain L1 of 33 humanized monoclonal antibody

SEQ ID NO: FR3 sequence of light chain L1 of 34 humanized monoclonal antibody

SEQ ID NO: FR4 sequence of 35 humanized monoclonal antibody light chain L1

SEQ ID NO:36 humanized monoclonal antibody heavy chain H3 nucleotide sequence

SEQ ID NO: FR1 sequence of light chain L2 of 42 humanized monoclonal antibody

SEQ ID NO: FR2 sequence of light chain L2 of 43 humanized monoclonal antibody

SEQ ID NO: FR3 sequence of light chain L2 of humanized monoclonal antibody 44

SEQ ID NO: FR4 sequence of light chain L2 of humanized monoclonal antibody 45

SEQ ID NO: FR1 sequence of 50 humanized monoclonal antibody light chain L3

SEQ ID NO: FR2 sequence of light chain L3 of 51 humanized monoclonal antibody

SEQ ID NO: FR3 sequence of light chain L3 of humanized monoclonal antibody 52

SEQ ID NO: FR4 sequence of 53 humanized monoclonal antibody light chain L3

The nucleotide sequences of the light chain and the heavy chain are connected into pcDNA3.1(+) vector plasmid based on seamless cloning to construct a recombinant expression vector. Wherein the combination of light and heavy chains is as follows: L1/H3, L2/H3 and L3/H3.

Expression and purification of the humanised engineered antibody plasmids in 293 cells was performed as in examples 1 ② and ③ 3 neutralizing antibodies were obtained, designated HZ-0412a, HZ-0412b and HZ-0412c respectively.

Example 7 determination of affinity of humanized antibody by enzyme-Linked immunosorbent assay (ELISA)

rhIL-6R-mFc antigen was diluted to 1. mu.g/ml with CB (pH 9.6), added to the microplate at 100. mu.l/well, and coated overnight at 4 ℃. After washing the plates 2 times with PBST, 200. mu.l/well of 3% BSA was added and blocked at 37 ℃ for 2 hours. The plate was washed 1 additional time with PBST, 100. mu.l/well of humanized antibody and Tocilizumab (Roche) at different concentrations (40. mu.g/ml start, 5-fold gradient dilution to 0.0001024. mu.g/ml) were added, respectively, and incubated at 37 ℃ for 2 hours. The plates were washed 3 times with PBST, and incubated for 1 hour at 37 ℃ with HRP (horseradish peroxidase) -labeled goat anti-human IgG antibody (ProteinTech, SA 00001-1). The plates were washed 4 times with PBST, 100. mu.l/well of TMB color developing solution (Zuman Bio, ZD311) was added, and after 15 minutes of incubation at 37 ℃ for color development, 50. mu.l/well of stop solution (1M sulfuric acid) was added, and absorbance was read at a wavelength of 450nm on a microplate reader (BioTek, ELx808) (see FIG. 2).

FIG. 2 shows that the affinity of each of the three monoclonal antibodies HZ-0412a, b and c for human IL-6R is significantly higher than that of Tocilizumab, with its EC₅₀The values are shown in table 1 below.

Name of antibody	EC₅₀(μg/mL)
		HZ-0412a	0.015
HZ-0412b	0.023
		HZ-0412c	0.026
Tocilizumab	0.031

Example 8 enzyme-linked immunosorbent assay (ELISA) of humanized antibodies inhibits the binding of IL-6 to IL-6R

gp130-His (RayBiotech, 230-30084) antigen was diluted to 4.0. mu.g/ml with PBS (pH 8.6), added to the plate at 100. mu.l/well, and coated overnight at 4 ℃. After 4 PBST washes, 300. mu.l/well of 3% BSA was added and blocked for 2 hours at 37 ℃. PBST 1 times of enzyme labeling plate washing, adding different concentrations of humanized antibody and Tocilizumab (Roche)50 u l/hole (60 u g/ml of the beginning, 3 times of gradient dilution to 0.0824 u g/ml), rhIL-6-hFc (by Beijing Virgiz and source biotechnology limited expression synthesis, nucleotide sequence of SEQ ID NO: 63, amino acid sequence of SEQ ID NO: 64) (8 u g/ml) and rhIL-6R-mFc (8 u g/ml) mixture 50 u l/hole, at 37 degrees C under the conditions of 1 hours of incubation. PBST was washed 4 times with the microplate, 100. mu.l/well of goat anti-mouse IgG antibody (ProteinTech, SA00001-1) labeled with HRP (horseradish peroxidase) was added, and incubated at 37 ℃ for 1 hour. The plates were washed 4 times with PBST, developed with 100. mu.l/well TMB developing solution (Zuman Bio, ZD311) by incubation at 37 ℃ for 15 minutes, developed with 50. mu.l/well stop solution (1M sulfuric acid), and absorbance was read at 450nm wavelength on a microplate reader (BioTek, ELx 808).

FIG. 3 shows that the inhibitory effect of three monoclonal antibodies HZ-0412a, HZ-0412b and HZ-0412c on the binding of IL-6/IL-6R complex to gp130 is significantly higher than that of Tocilizumab. Indicating that HZ-0412a, b and c act by inhibiting the IL-6/IL-6R/gp130 ternary complex, while Tocilizumab acts by inhibiting the IL-6/IL-6R binary complex.

Example 9 humanized monoclonal antibody inhibits proliferation of DS-1 cells

(10% fetal bovine serum in RPMI-1640 Medium + Hepes + sodium pyruvate) Medium Dilute DS-1 cells

CRL-11102^TM) To 4X 10⁴～8×10⁴Perml, 100. mu.l/well of 96-well plate was inoculated and starved for 24 hours. Humanized antibody and Tocilizumab (Roche) (50. mu.g/ml start, 5-fold gradient dilution to 2.56E-05. mu.g/ml) were diluted with a (10% fetal bovine serum in RPMI-1640 medium + -Hepes + sodium pyruvate +20ng/ml IL-6) gradient and 100. mu.l DS-1 cells were added per well. Culturing for 72-96 hours, adding 10 mul CCK-8 reagent, developing for 2-4 hours, and measuring absorbance value by A450.

The absorbance at 450nm is in a linear relation with the number of living cells in the culture medium, the number of the living cells can be determined by using a corresponding absorbance value and a standard curve, and the experimental result is shown in figure 4, and the concentrations of the three humanized antibodies and the Tocilizumab are in negative correlation with the number of the living cells in the range of 0-50 mu g/ml, so that the proliferation of DS-1 cells can be inhibited. In a low concentration range (0.00064-2 mu g/ml), the three humanized antibodies HZ-0412a, b and c have stronger inhibitory effect than Tocilizumab.

Example 10 inhibition of IL-6 stimulated phosphorylation of STAT-3 by humanized antibodies on DLD-1 cells

Humanized antibody and Tocilizumab (0.2, 0.4, 1 and 5 μ g/ml) at the same concentration were added to DLD-1 cells, respectively

CCL-221^TMAnd incubating at 37 ℃ for 2 hours, adding 10ng/ml rhIL-6-His into each well, incubating for 30min, removing culture medium supernatant, washing for 3 times by PBS, adding RIPA lysate to lyse cells, and collecting protein. Collecting the protein by SDS-PAAfter GE electrophoresis, Western Blot detects the STAT-3 phosphorylation inhibition level of the humanized antibody on the DLD-1 cells stimulated by IL-6.

Further, a concentration of humanized antibody (3.3333 μ g/ml start, 3-fold gradient diluted to 0.0015 μ g/ml) and Tocilizumab (30 μ g/ml start, 3-fold gradient diluted to 0.0137 μ g/ml) were tested for phosphorylation levels of p-STAT3(Tyr705) (Cell Signaling, 52075) and total STAT3 by ELISA assay as described above.

As shown in FIGS. 5 and 6, the neutralizing activity of the 3-strain IL-6R humanized antibody was superior to that of Tocilizumab as seen from the Western Blot; the neutralization activity shows a gradient effect; according to the gray value preliminary analysis experiment result: of the 3 humanized antibodies, HZ-0412a showed the best inhibitory effect, about 100 times that of Tocilizumab, while the other two humanized antibodies were slightly weaker, about 20 times that of Tocilizumab.

Furthermore, as can be seen from the ELISA result shown in FIG. 7, the humanized antibodies HZ-0412a, HZ-0412b and HZ-0412c of the 3 strains are all stronger than Tocilizumab, wherein the HZ-0412a and HZ-0412b have better effect and the inhibition effect is about 5-10 times that of Tocilizumab.

The results of FIG. 5-FIG. 7 show that 3 humanized antibodies HZ-0412a, HZ-0412b and HZ-0412c can prevent IL-6 from binding with receptor IL-6R, inhibit gp130 signal transduction, reduce the phosphorylation level of downstream signal protein p-Stat3(Tyr705), and have stronger inhibition effect than Tocilizumab.

Example 11 humanized antibodies inhibit SAA secretion from rhIL-6 stimulated HepG2 cells

Human liver cancer cell HepG2 (basic medicine cell center of institute of basic medicine of Chinese academy of medical sciences, 3111C0001CCC000802) at 2X 10⁵Cells/well were plated into 48 well plates and incubated in MEM NEAA medium (Thermo, 41500034) for approximately 24 hours, a concentration of humanized antibody and Tocilizumab (50. mu.g/mL start, 3 fold gradient dilution to 0.0025. mu.g/mL) were mixed with 100ng/mL rhIL-6-His and 200ng/mL rhIL-6R (ProtoHotan, 10398-H02H) and incubated at 37 ℃ for 2 hours, 25ng/mL IL-1 β (ProtoHotan, 10139-HNAE) was added and mixed well, HepG2 cells were added to the mixture and incubated for 48 hours, and the supernatant of the culture was collected using ELISA kit (R. RTM. kit (R. RTM. ProtoHotan. Shen, 10139-HNAE)&D，DY3019-05)The SAA in the supernatant was measured.

As shown in FIG. 8, the three humanized antibodies HZ-0412a, HZ-0412b, HZ-0412c and Tocilizumab were all able to inhibit the SAA secretion of rhIL-6-stimulated HepG2 cells in a concentration-dependent manner. The neutralizing effect of HZ-0412a is slightly better than that of Tocilizumab, and the neutralizing activity of HZ-0412b and HZ-0412c is equivalent to that of Tocilizumab.

Example 12 determination of affinity constant of humanized antibody

ForteBio Blitz biomolecular interaction assay (ForteBio) instrument measures the affinity of HZ-0412a, HZ-0412b and HZ-0412c, and Tocilizumab for human IL-6R. The affinity constants determined are shown in Table 2 below.

Name of antibody	KD(M)	Ka(1/Ms)	kd(1/s)
				HZ-0412a	8.205E-9	1.403E4	1.151E-4
HZ-0412b	1.957E-8	1.801E4	3.526E-4
				HZ-0412c	2.026E-8	8.890E3	1.801E-4
Tocilizumab	9.825E-8	3.516E3	3.454E-4

Example 13 Cross-reactivity of humanized antibodies with rat monkey and human IL-6R

Rhesus IL-6R (Yi Qiao Shen, 90197-CNAE) and rat IL-6R (Yi Qiao Shen, 80076-RNAE) were diluted to 2. mu.g/ml with CB, added to the microplate at 100. mu.l/well, and coated overnight at 4 ℃. After washing the plates 2 times with PBST, 300. mu.l/well of 3% BSA was added and blocked at 37 ℃ for 2 hours. The plate was washed 1 more times with PBST, and 100. mu.l/well of humanized antibody (40. mu.g/ml starting, 5-fold gradient diluted to 0.0001024. mu.g/ml) was added and incubated at 37 ℃ for 2 hours. The plates were washed 3 times with PBST, and incubated for 1 hour at 37 ℃ with HRP (horseradish peroxidase) -labeled goat anti-human IgG antibody (Proteintetech, SA 00001-1). The plates were washed 4 times with PBST, added with 100. mu.l/well of TMB developing solution (Zuman Bio, ZD311), incubated at 37 ℃ for 15 minutes for development, and the absorbance was read at 450nm on a microplate reader (Bio-Rad, Model 680 Micro reader).

The results are shown in FIG. 9 (left panel: cross-reactivity of humanized antibody to rhesus IL-6R; right panel: cross-reactivity of humanized antibody to rat IL-6R). The affinity values of the humanized antibodies for rhesus IL-6R were more similar compared to the affinity values of the humanized antibodies for human IL-6R.

Claims

1. An antibody or antigen-binding fragment thereof that binds human IL-6R, wherein the antibody comprises:

HCDR1 consisting of the sequence shown in SEQ ID NO. 6,

HCDR2 consisting of the sequence shown in SEQ ID NO:7, and

HCDR3 consisting of the sequence shown in SEQ ID NO. 8,

and the antibody further comprises:

LCDR1 consisting of the amino acid sequence shown in SEQ ID NO. 9,

LCDR2 consisting of the amino acid sequence shown in SEQ ID NO:10, and

LCDR3 consisting of the sequence shown in SEQ ID NO. 11,

wherein the antigen binding fragment is selected from the group consisting of Fab, Fab ', F (ab')₂Fv, Fab/c, single-chain antibody or diabody.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody comprises:

(1) (i) a heavy chain variable region consisting of the sequence:

the amino acid sequence shown as SEQ ID NO. 4, or

A sequence having from 93% to less than 100% sequence identity to the sequence shown in SEQ ID NO. 4, and

(ii) a light chain variable region consisting of the sequence:

the amino acid sequence shown as SEQ ID NO. 5, or

A sequence having 80% or more to less than 100% sequence identity to the sequence shown in SEQ ID NO. 5;

(2) (i) a heavy chain variable region comprising or consisting of the sequence:

the amino acid sequence shown as SEQ ID NO. 26, or

A sequence having more than or equal to 93-less than 100% sequence identity with the sequence shown in SEQ ID NO. 26

And are and

(ii) a light chain variable region comprising or consisting of the sequence:

the amino acid sequence shown as SEQ ID NO. 27, or

A sequence having from 85% to less than 100% column identity to the sequence set forth in SEQ ID NO 27;

(3) (i) a heavy chain variable region comprising or consisting of the sequence:

the amino acid sequence shown as SEQ ID NO. 26, or

A sequence having from 93% to less than 100% sequence identity to the sequence shown in SEQ ID NO 26, and

(ii) a light chain variable region comprising or consisting of the sequence:

the amino acid sequence shown as SEQ ID NO. 41, or

A sequence having from 86% to less than 100% sequence identity to the sequence shown as SEQ ID NO. 41; or

(4) (i) a heavy chain variable region comprising or consisting of the sequence:

the amino acid sequence shown as SEQ ID NO. 26, or

(ii) a light chain variable region comprising or consisting of the sequence:

the amino acid sequence shown as SEQ ID NO. 49, or

A sequence having greater than or equal to 80% to less than 100% sequence identity to the sequence shown in SEQ ID NO. 49.

3. A polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1-2, wherein the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof are each encoded by the following nucleotide sequences:

(1) (i) the nucleotide sequence shown as SEQ ID NO:22, or

A sequence having from 93% to less than 100% sequence identity to the sequence shown in SEQ ID NO. 22, and

(ii) the nucleotide sequence shown as SEQ ID NO. 23, or

A sequence having greater than or equal to 80% to less than 100% sequence identity to the sequence set forth in SEQ ID NO. 23;

(2) (i) the nucleotide sequence shown as SEQ ID NO:38, or

A sequence having from 93% to less than 100% sequence identity to the sequence shown in SEQ ID NO 38, and

(ii) the nucleotide sequence shown as SEQ ID NO. 39, or

A sequence having from 85% to less than 100% sequence identity to the sequence set forth in SEQ ID NO. 39;

(3) (i) the nucleotide sequence shown as SEQ ID NO:38, or

(ii) the nucleotide sequence shown as SEQ ID NO. 47, or

A sequence having from 86% to less than 100% sequence identity to the sequence set forth in SEQ ID NO. 47; or

(4) (i) the nucleotide sequence shown as SEQ ID NO:38, or

(ii) the nucleotide sequence shown as SEQ ID NO. 55, or

A sequence having more than or equal to 80% to less than 100% sequence identity to the sequence shown in SEQ ID NO. 55.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody further comprises the framework regions FR-H1, FR-H2, FR-H3, and FR-H4 of the heavy chain variable region, and the framework regions FR-L1, FR-L2, FR-L3, and FR-L4 of the light chain variable region, wherein

(1) FR-H1 consists of the amino acid sequence of SEQ ID NO. 12;

FR-H2 consists of the amino acid sequence of SEQ ID NO 13;

FR-H3 consists of the amino acid sequence of SEQ ID NO. 14;

FR-H4 consists of the amino acid sequence of SEQ ID NO. 15; and

FR-L1 consists of the amino acid sequence of SEQ ID NO. 16; FR-L2 consists of the amino acid sequence of SEQ ID NO 17; FR-L3 consists of the amino acid sequence of SEQ ID NO. 18; FR-L4 consists of the amino acid sequence of SEQ ID NO. 19;

(2) FR-H1 consists of the amino acid sequence of SEQ ID NO 28;

FR-H2 consists of the amino acid sequence of SEQ ID NO: 29;

FR-H3 consists of the amino acid sequence of SEQ ID NO 30;

FR-H4 consists of the amino acid sequence of SEQ ID NO: 31; and

FR-L1 consists of the amino acid sequence of SEQ ID NO: 32;

FR-L2 consists of the amino acid sequence of SEQ ID NO. 33;

FR-L3 consists of the amino acid sequence of SEQ ID NO: 34;

FR-L4 consists of the amino acid sequence of SEQ ID NO: 34;

(3) FR-H1 consists of the amino acid sequence of SEQ ID NO 28;

FR-H2 consists of the amino acid sequence of SEQ ID NO: 29;

FR-H3 consists of the amino acid sequence of SEQ ID NO 30;

FR-H4 consists of the amino acid sequence of SEQ ID NO: 31; and

FR-L1 consists of the amino acid sequence of SEQ ID NO: 42;

FR-L2 consists of the amino acid sequence of SEQ ID NO: 43;

FR-L3 consists of the amino acid sequence of SEQ ID NO: 44;

FR-L4 consists of the amino acid sequence of SEQ ID NO: 45; or

(4) FR-H1 consists of the amino acid sequence of SEQ ID NO 28;

FR-H2 consists of the amino acid sequence of SEQ ID NO: 29;

FR-H3 consists of the amino acid sequence of SEQ ID NO 30;

FR-H4 consists of the amino acid sequence of SEQ ID NO:31, and

FR-L1 consists of the amino acid sequence of SEQ ID NO: 50;

FR-L2 consists of the amino acid sequence of SEQ ID NO: 51;

FR-L3 consists of the amino acid sequence of SEQ ID NO. 52;

FR-L4 consists of the amino acid sequence of SEQ ID NO: 53.

5. The antibody or antigen-binding fragment thereof of any one of claims 1, 2 or 4, wherein the antibody consists of an amino acid sequence selected from the group consisting of the following heavy and light chain combinations:

(1) the amino acid sequence of SEQ ID NO. 2 and the amino acid sequence of SEQ ID NO. 3;

(2) the amino acid sequence of SE ID NO. 24 and the amino acid sequence of SEQ ID NO. 25;

(3) the amino acid sequence of SEQ ID NO. 24 and the amino acid sequence of SEQ ID NO. 40, or

(4) The amino acid sequence of SEQ ID NO. 24 and the amino acid sequence of SEQ ID NO. 48.

6. A polynucleotide encoding the antibody or antigen-binding fragment thereof of claim 5, wherein the heavy and light chains of the antibody or antigen-binding fragment thereof are each encoded by a nucleotide sequence of SEQ ID NO:

(1) the nucleotide sequence of SEQ ID NO. 20 and the nucleotide sequence of SEQ ID NO. 21;

(2) the nucleotide sequence of SE ID NO. 36 and the nucleotide sequence of SEQ ID NO. 37;

(3) the nucleotide sequence of SEQ ID NO. 36 and the nucleotide sequence of SEQ ID NO. 46; or

(4) The nucleotide sequence of SEQ ID NO. 36 and the nucleotide sequence of SEQ ID NO. 54.

7. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a humanized antibody, a chimeric antibody, or a multispecific antibody.

8. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a bispecific antibody.

9. The antibody or antigen-binding fragment thereof of claim 7, wherein the constant region of the antibody is humanized.

10. The antibody or antigen-binding fragment thereof of claim 9, wherein the constant region of the antibody is from a human IgG.

11. The antibody or antigen-binding fragment thereof of claim 9, wherein the constant region of the antibody is from human IgG1 or IgG 4.

12. The antibody or antigen-binding fragment thereof of claim 9, wherein the heavy chain constant region of the antibody is represented by Iggamma-1 or Ig gamma-4 chain C region; the light chain constant region adopts an Ig kappa chain C region.

13. The antibody or antigen-binding fragment thereof of claim 12, wherein the heavy chain constant region of the antibody is an Iggamma-1 chain C region and the light chain constant region is an Ig kappa chain C region with GenBank ACCESSION No. access: P01834.

14. A vector comprising the polynucleotide of claim 3 or 6.

15. A host cell comprising the polynucleotide of claim 3 or 6 or the vector of claim 14.

16. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-2,4-13, comprising culturing the host cell of claim 15.

17. An antibody conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-2,4-13 and a conjugate moiety conjugated thereto.

18. The antibody conjugate of claim 17, wherein the conjugate moiety is selected from the group consisting of a purification tag, a cytotoxic agent, a radioisotope, a chemiluminescent substance, an enzyme, and polyethylene glycol.

19. The antibody conjugate of claim 18, wherein the purification tag is a His tag.

20. A multispecific antibody comprising the antibody or antigen-binding fragment thereof of any one of claims 1-2,4-13, and an antibody or antigen-binding fragment directed to another antigen and/or other epitope.

21. The multispecific antibody of claim 20, which is a bispecific antibody.

22. A fusion protein comprising the antibody or antigen-binding fragment thereof of any one of claims 1-2, 4-13.

23. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-2,4-13, the antigen conjugate of any one of claims 18-19, the multispecific antibody of claim 20 or 21, or the fusion protein of claim 22.

24. The pharmaceutical composition of claim 23, further comprising a pharmaceutically acceptable carrier and/or excipient.

25. The pharmaceutical composition of claim 23 or 24, which is in a dosage form suitable for administration by injection.

26. The pharmaceutical composition of claim 25, which is in a dosage form suitable for intravenous injection.

27. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-2,4-13, the antigen conjugate of any one of claims 18-19, the multispecific antibody of claim 20 or 21, or the fusion protein of claim 22.

28. The kit of claim 27, further comprising a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof of any one of claims 1-2,4-13, the antigen conjugate of any one of claims 18-19, the multispecific antibody of claim 20 or 21, or the fusion protein of claim 22.

29. The kit of claim 28, wherein the second antibody further comprises a radioisotope, a chemiluminescent substance, an enzyme.

30. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-2,4-13, the antigen conjugate of any one of claims 18-19, the multispecific antibody of claim 20 or 21, or the fusion protein of claim 22 in the preparation of a kit for detecting the presence or level of human IL-6R in a sample.

31. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-2,4-13, the antigen conjugate according to any one of claims 18-19, the multispecific antibody according to claim 20 or 21 or the fusion protein according to claim 22 for the manufacture of a medicament for the prevention and/or treatment and/or co-treatment and/or diagnosis of multicentric Castleman disease, rheumatoid arthritis, childhood rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, multiple myeloma.

32. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-2,4-13, the antigen conjugate according to any one of claims 18-19, the multispecific antibody according to claim 20 or 21, or the fusion protein according to claim 22 for the manufacture of a medicament for the adjunctive treatment and/or diagnosis of multicentric Castleman disease, rheumatoid arthritis, childhood rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, multiple myeloma.