CN115746132B

CN115746132B - anti-IL-17A antibodies and uses thereof

Info

Publication number: CN115746132B
Application number: CN202111031745.0A
Authority: CN
Inventors: 刘婵娟; 郎国竣; 张文海; 周蕴华; 胡宇豪; 闫闰; 孙兴鲁; 雷攀
Original assignee: Sanyou Biopharmaceuticals Co Ltd
Current assignee: Sanyou Biopharmaceuticals Co Ltd
Priority date: 2021-09-03
Filing date: 2021-09-03
Publication date: 2023-09-08
Anticipated expiration: 2041-09-03
Also published as: CN115746132A; WO2023030480A1

Abstract

The invention belongs to the field of biological medicine. In particular, the invention relates to antibodies or antigen-binding fragments thereof that specifically bind to IL-17A, methods of making, and uses thereof.

Description

anti-IL-17A antibodies and uses thereof

Technical Field

Background

IL-17 (interleukin-17) family cytokines comprise 6 members, IL-17A, IL-17B, IL-17C, IL-17D, IL-17E and IL-17F, respectively; the most representative member of the IL-17 family is IL-17A. IL-17A was first found in the production of T cells in 1993 and was expressed in a variety of T cells. The human IL-17A gene comprises 155 amino acids, a signal peptide of 19 amino acids and a mature region of 132 amino acids, has a relative molecular mass of 17kDa, and exists as an IL-17A homodimer or an IL-17A/IL-17F heterodimer (Gaffen Sarah L, nat. Rev. Immunol.,2009, 9:556-67).

When the human body is injured or damaged, lymphocytes migrating to the infected or damaged area of the body secrete IL-17A. IL-17A induces the expression of inflammatory factors and chemokines on the one hand, thereby recruiting more immune cells to the site of inflammation to exacerbate the inflammatory response; on the other hand, IL-17A also induces the expression of some tissue repair related factors, thereby accelerating the recovery of the body. Although IL-17A plays a role in expanding immune defenses and protecting the body during host anti-infection and tissue repair, IL-17A is highly expressed in many autoimmune patients. Excessive IL-17A levels induce the expression of a number of inflammatory factors, which contribute to the progression of autoimmune diseases. A number of animal models demonstrate that the absence of IL-17A or the neutralization of IL-17A by antibodies can effectively inhibit the pathological extent of a variety of autoimmune diseases. Evidence suggests that the treatment of autoimmune diseases including Rheumatoid Arthritis (RA), psoriasis, crohn's disease, multiple Sclerosis (MS), asthma, lupus erythematosus, etc. with IL-17A signaling pathway as a target has certain therapeutic effects.

Among them, the clinical evaluation of the therapeutic effect of psoriasis generally uses the skin lesion area and severity index PASI (psoriasis area and severity index), and specifically PASI 75, PASI 90 and PASI 100 as important indicators of the success of the treatment. Currently, two monoclonal antibodies, secukinumab (Stuzumab) and ixekizumab, directed against IL-17A and one monoclonal antibody, brodalumab, directed against IL-17RA have been used in clinical batches for the treatment of moderate to severe plaque psoriasis. Although these three monoclonal antibodies have been reported to be more than 80% effective in PASI 75 for moderate to severe plaque psoriasis, PASI 100 has no more than 40% (April W. Armstrong et al, JAMA 2020;323 (19): 1945-1960). Therefore, in order to further improve the therapeutic effects on disorders such as psoriasis, it is necessary to continue to develop new drugs targeting IL-17A with better therapeutic effects.

Disclosure of Invention

In one aspect, the invention provides an anti-IL-17A antibody or antigen-binding fragment thereof comprising a single variable domain of an immunoglobulin that is a first family, second family, or third family single variable domain.

(a) First family single variable domains

In some embodiments, the first family single variable domain comprises:

1) CDR1 comprising the amino acid sequence of SEQ ID NO: 68:

Xaa1-Xaa2-Xaa3-Xaa4-S-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9(SEQ ID NO:68)

wherein, the liquid crystal display device comprises a liquid crystal display device,

xaa1 is G or A; xaa2 is F or S; xaa3 is T or I; xaa4 is F or I; xaa5 is S, D or I; xaa6 is Y, F or H; xaa7 is A or P; xaa8 is M or I; xaa9 is S, G or A;

2) CDR2 comprising the amino acid sequence of SEQ ID NO: 69:

Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-S-T-Xaa17(SEQ ID NO:69)

xaa10 is T or A; xaa11 is V or I; xaa12 is E, T or absent; xaa13 is I, S or T; xaa14 is G, A, R or N; xaa15 is G, S or V; xaa16 is S or G; xaa17 is N or D; and

3) CDR3 comprising the amino acid sequence of SEQ ID No. 70:

D-Xaa18-Xaa19-Xaa20-Y-E-Xaa21-Xaa22-D-D-Y(SEQ ID NO:70)

xaa18 is W, Y or G; xaa19 is K, T or R; xaa20 is W or Y; xaa21 is V, S or H; xaa22 is I or absent.

In a preferred embodiment, the first family single variable domain comprises:

1) CDR1 comprising the amino acid sequence of SEQ ID NO 17, 38, 20, 23 or 35;

2) CDR2 comprising the amino acid sequence of SEQ ID NO. 18, 39, 21, 24 or 36; and

3) CDR3 comprising the amino acid sequence of SEQ ID NO. 19, 40, 22, 25 or 37.

In a specific embodiment, the first family single variable domain comprises:

(1) CDR1 comprising the amino acid sequence of SEQ ID NO. 17; CDR2 comprising the amino acid sequence of SEQ ID NO. 18; and CDR3 comprising the amino acid sequence of SEQ ID NO. 19; or alternatively

(2) CDR1 comprising the amino acid sequence of SEQ ID NO. 38; CDR2 comprising the amino acid sequence of SEQ ID NO 39; and CDR3 comprising the amino acid sequence of SEQ ID NO. 40; or alternatively

(3) CDR1 comprising the amino acid sequence of SEQ ID NO. 20; CDR2 comprising the amino acid sequence of SEQ ID NO. 21; and CDR3 comprising the amino acid sequence of SEQ ID NO. 22; or alternatively

(4) CDR1 comprising the amino acid sequence of SEQ ID NO. 23; CDR2 comprising the amino acid sequence of SEQ ID NO. 24; and CDR3 comprising the amino acid sequence of SEQ ID NO. 25; or alternatively

(5) CDR1 comprising the amino acid sequence of SEQ ID NO. 35; CDR2 comprising the amino acid sequence of SEQ ID NO. 36; and CDR3 comprising the amino acid sequence of SEQ ID NO. 37 or a variant thereof.

(b) Second family single variable domains

In some embodiments, the second family single variable domain comprises:

1) CDR1 comprising the amino acid sequence of SEQ ID NO: 71:

Xaa23-Xaa24-I-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-M-Xaa30(SEQ ID NO:71)

xaa23 is A or G; xaa24 is S or F; xaa25 is F or I; xaa26 is N or S; xaa27 is A, I or E; xaa28 is H or Y; xaa29 is A or S; xaa30 is G or N;

2) CDR2 comprising the amino acid sequence of SEQ ID NO: 72:

Xaa31-I-T-Xaa32-G-G-Xaa33-T-Xaa34(SEQ ID NO:72)

xaa31 is A, S, T or R; xaa32 is S, Y or R; xaa33 is S, N or T; xaa34 is D or N; and

3) CDR3 comprising the amino acid sequence of SEQ ID NO. 28, 31, 34 or 43.

In a preferred embodiment, the second family single variable domain comprises:

1) CDR1 comprising the amino acid sequence of SEQ ID NO. 26, 29, 32 or 41;

2) CDR2 comprising the amino acid sequence of SEQ ID NO 27, 30, 33 or 42; and

3) CDR3 comprising the amino acid sequence of SEQ ID NO. 28, 31, 34 or 43.

In a specific embodiment, the second family single variable domain comprises:

(1) CDR1 comprising the amino acid sequence of SEQ ID NO. 26; CDR2 comprising the amino acid sequence of SEQ ID NO. 27; and CDR3 comprising the amino acid sequence of SEQ ID NO. 28; or alternatively

(2) CDR1 comprising the amino acid sequence of SEQ ID NO. 29; CDR2 comprising the amino acid sequence of SEQ ID NO. 30; and CDR3 comprising the amino acid sequence of SEQ ID NO. 31; or alternatively

(3) CDR1 comprising the amino acid sequence of SEQ ID NO. 32; CDR2 comprising the amino acid sequence of SEQ ID NO. 33; and CDR3 comprising the amino acid sequence of SEQ ID NO. 34; or alternatively

(4) CDR1 comprising the amino acid sequence of SEQ ID NO. 41; CDR2 comprising the amino acid sequence of SEQ ID NO. 42; and CDR3 comprising the amino acid sequence of SEQ ID NO. 43.

(c) Third family single variable domains

In some embodiments, the third family single variable domain comprises:

1) CDR1 comprising the amino acid sequence of SEQ ID NO: 73:

G-F-Xaa35-L-D-Xaa36-Xaa37-Xaa38-I-G(SEQ ID NO:73)

xaa35 is T or N; xaa36 is D or Y; xaa37 is D or Y; xaa38 is A or G;

2) CDR2 comprising the amino acid sequence of SEQ ID No. 74:

C-I-Xaa39-S-S-D-G-S-T-Y(SEQ ID NO:74)

xaa39 is S or T; and

3) CDR3 comprising the amino acid sequence of SEQ ID NO 46, 49 or 52.

In a preferred embodiment, the third family single variable domain comprises:

1) CDR1 comprising the amino acid sequence of SEQ ID NO 44, 47 or 50;

2) CDR2 comprising the amino acid sequence of SEQ ID NO 45, 48 or 51; and

3) CDR3 comprising the amino acid sequence of SEQ ID NO 46, 49 or 52.

In a specific embodiment, the third family single variable domain comprises:

(1) CDR1 comprising the amino acid sequence of SEQ ID NO. 44; CDR2 comprising the amino acid sequence of SEQ ID NO. 45; and CDR3 comprising the amino acid sequence of SEQ ID NO. 46; or alternatively

(2) CDR1 comprising the amino acid sequence of SEQ ID NO. 47; CDR2 comprising the amino acid sequence of SEQ ID NO. 48; and CDR3 comprising the amino acid sequence of SEQ ID NO. 49; or alternatively

(3) CDR1 comprising the amino acid sequence of SEQ ID NO. 50; CDR2 comprising the amino acid sequence of SEQ ID NO. 51; and CDR3 comprising the amino acid sequence of SEQ ID NO. 52.

In one embodiment, the single variable domain comprises an amino acid sequence selected from the group consisting of: SEQ ID NO. 5, SEQ ID NO. 12, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16.

In one embodiment, the single variable domain comprises an amino acid sequence selected from the group consisting of: SEQ ID NO. 65, SEQ ID NO. 67 and SEQ ID NO. 66.

In some embodiments, the anti-IL-17A antibody or antigen-binding fragment thereof is conjugated to at least one detectable label.

In another aspect, the invention also provides a multispecific antibody comprising a first antigen-binding portion which binds IL-17A and a second antigen-binding portion which binds a second antigen, wherein the first antigen-binding portion comprises an anti-IL-17A antibody or antigen-binding fragment thereof of the invention.

In yet another aspect, the invention provides a polynucleotide encoding an anti-IL-17A antibody or antigen-binding fragment thereof of the invention.

The invention also provides an expression vector comprising a polynucleotide of the invention.

In another aspect, the invention also provides a host cell comprising a polynucleotide or expression vector of the invention.

The invention also provides a method of making an anti-IL-17A antibody or antigen-binding fragment thereof of the invention, comprising culturing a host cell of the invention under suitable conditions to express the antibody or antigen-binding fragment thereof, and isolating the antibody or antigen-binding fragment thereof from the host cell or culture thereof.

In yet another aspect, the invention also provides a pharmaceutical composition comprising an anti-IL-17A antibody or antigen-binding fragment thereof of the invention, and a pharmaceutically acceptable carrier.

The invention also provides the use of an anti-IL-17A antibody or antigen-binding fragment thereof or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of an IL-17A mediated disease.

The invention also provides a method of detecting the presence or determining the expression level of IL-17A in a sample comprising:

(a) Contacting an anti-IL-17A antibody or antigen-binding fragment thereof of the invention with the sample, and

(b) Detecting the formation of or determining the amount of an immune complex between the antibody or antigen binding fragment thereof and IL-17A in the sample, thereby detecting the presence of IL-17A in the sample or determining the expression level of IL-17A in the sample.

Drawings

FIGS. 1A-1C show the binding activity of anti-IL-17A nanobodies to recombinant antigen IL-17A-His.

FIGS. 2A-2C show the blocking activity of anti-IL-17A nanobodies against IL-17A and IL-17 RA.

FIGS. 3A-3B show SDS-PAGE of anti-IL-17A heavy chain antibodies under non-denaturing and denaturing conditions.

FIGS. 4A-4C show the binding activity of an anti-IL-17A heavy chain antibody to recombinant antigen IL-17A.

FIGS. 5A-5C show the blocking activity of anti-IL-17A heavy chain antibodies against IL-17A/IL-17 RA.

FIGS. 6A-6K show the ability of anti-IL-17A heavy chain antibodies to neutralize IL-17A-induced secretion of IL-6 by HeLa cells.

FIGS. 7A-7B show the binding activity of an anti-IL-17A humanized antibody to recombinant antigen IL-17A.

Detailed Description

Definition of the definition

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures as used herein are terms and conventional procedures that are widely used in the corresponding arts. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

Herein, immunoglobulins include any immunoglobulin type (e.g., igG, igM, igD, igE, igA and IgY), any class (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2), or subclass (e.g., igG2a and IgG2 b).

As used herein, "antibody" refers to an immunoglobulin or fragment thereof that specifically binds an epitope through at least one antigen binding site. Herein, the definition of antibody encompasses antigen binding fragments. The term "antibody" includes multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, heavy chain antibodies, single domain antibodies, and antigen-binding fragments. Antibodies may be synthetic (e.g., produced by chemical or biological coupling), enzymatically treated, or recombinantly produced.

As used herein, an "antigen binding fragment" refers to a portion of a full-length antibody that is less than full length, but that comprises at least a portion of the variable region of the full-length antibody (e.g., comprises one or more CDRs and/or one or more antigen binding sites), and thus retains at least a portion of the ability of the full-length antibody to specifically bind an antigen.

As used herein, "immunoglobulin single variable domain" or "single variable domain" refers to a single variable region (variable domain) having antigen binding activity. Unlike conventional antibodies, which consist of a pair of VH and VL functional antigen-binding units, a single variable domain can alone form a functional antigen-binding unit. The single variable domains may be derived from naturally occurring light chain-free antibodies, such as the variable domains (variable domain of heavy chain of heavy-chain antibodies, VHH) of heavy chain antibodies of camelids (e.g., camels and alpacas) and the single variable domains (IgNAR variable single-domain, VNAR) of the shark's neoantigen receptor, or may be selected from full length antibodies, such as the light chain variable domains and heavy chain variable domains of human antibodies that have antigen binding activity. VHHs typically comprise three highly variable "Complementarity Determining Regions (CDRs)" and four relatively conserved "Framework Regions (FRs)" and are connected in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from N-terminus to C-terminus.

As used herein, "single domain antibody (sdAb)" or "nanobody" refers to an antibody that comprises a single immunoglobulin variable domain as a functional antigen-binding unit. Unlike full length antibodies, which typically comprise two heavy and two light chains, single domain antibodies typically comprise a single peptide chain consisting of a single variable domain, with a molecular weight of only about 15 kDa.

As used herein, the terms "Heavy-chain-only antibody" and "Heavy-chain antibody" are used interchangeably and are presented in their broadest sense to refer to antibodies lacking the light chain of a conventional antibody that comprise only one VHH and do not comprise the Heavy chain constant region of CH1 (e.g., an Fc fragment).

The CDRs can be identified by those skilled in the art using methods well known in the art, for example using Kabat, abM, chothia or IMGT numbering. In this context, the CDRs of the anti-IL-17A antibodies of the invention are defined in terms of the AbM numbering system, but it will be appreciated by those skilled in the art that multiple CDR numbering systems may also be used for the same variable region, such as Chothia, kabat and IMGT. Although the CDR boundaries defined by the different numbering systems may be different, the CDRs corresponding to the same numbering system represent potent antigens capable of binding to an epitope of an antigen Binding sites. For a description of CDR numbering systems see for example,kabat numbering system：Kabat，E.A.et al.(1991)Sequences of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242；Chothia numbering system：Chothia,C.et al.(1987)J.Mol.Biol.196:901-917；IMGT numbering system: lefranc, m. -p.,2011 (6), IMGT, the International ImMunoGeneTics Information System Cold Spring Harb protoc, and Lefranc, m. -p.et., dev.comp.immunol.,27,55-77 (2003);AbM numbering system：Martin,A.C.R.and J.Allen(2007)“Bioinformatics tools for antibody engineering，”in S.Dübel(ed.)，Handbook of Therapeutic Antibodies.Weinheim:Wiley-VCH Verlag，pp.95–118。

As used herein, the terms "framework region" and "framework region" are used interchangeably. As used herein, the term "framework region", "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR sequences as defined above.

"Fc fragment" generally refers to a crystallizable fragment of a conventional antibody or heavy chain antibody that has been digested with papain. In general, the Fc fragment of IgG and heavy chain antibodies may comprise part of the hinge region, CH2 and CH3. In this context, the Fc fragment may comprise at least part of the hinge region (e.g., all or part of the hinge region), CH2 and CH3. "hinge region" generally refers to the portion between CH1 and CH2 in a conventional antibody, the portion between VHH and CH2 in a heavy chain antibody, or a functional equivalent thereof, such as the hinge region in a T Cell Receptor (TCR). The hinge region may be a complete hinge region or a portion thereof. Those skilled in the art can determine CDR, FR, VH, VL, CL, CH, CH2, CH3 and the location of the hinge region in the antibody based on known algorithms and software, and a description of applicable algorithms and software can be found, for example, in William r.strohl, lila m.strohl, (2012), antibody structure-function relationships, in Woodhead Publishing Series in Biomedicine, therapeutic Antibody Engineering, woodhead Publishing, pp.37-56.

As used herein, "chimeric antibody" refers to an antibody in which a portion (e.g., CDR, FR, variable region, constant region, or a combination thereof) is identical or homologous to a corresponding sequence in an antibody derived from a particular species, and the remainder is identical or homologous to a corresponding sequence in an antibody derived from another species. In some embodiments of the invention, the chimeric antibody comprises variable regions derived from non-human species (e.g., camelids, such as camels and alpacas) and constant regions derived from different species (e.g., humans). Chimeric antibodies may also refer to multispecific antibodies that are specific for at least two different antigens. Chimeric antibodies may be produced by antibody engineering. Methods of antibody engineering are well known to those skilled in the art. In particular, chimeric antibodies can be generated by DNA recombinant techniques (see, e.g., sambrook, j., et al (1989). Molecular cloning: a laboratory manual,2nd ed.Cold Spring Harbor Laboratory,Cold Spring Harbor,N.Y).

As used herein, the term "humanized antibody" refers to an antibody in which a non-human antibody is modified to increase sequence homology with a human antibody. Humanized antibodies generally retain the antigen binding capacity of the non-human antibody from which they are derived and have low immunogenicity to humans. Humanized antibodies may be obtained by antibody engineering any non-human species antibody or antibodies (e.g., chimeric antibodies) comprising sequences derived from a non-human species therein. Techniques for obtaining humanized antibodies from non-human antibodies are well known to those skilled in the art, for example, CDR sequences of non-human antibodies (e.g., camelid antibodies) may be grafted into human antibody framework regions. In some cases, to preserve the antigen binding capacity and/or stability of a humanized antibody, critical amino acid residues of a non-human antibody (e.g., camelid antibody) framework sequence may be retained in the human antibody framework region, i.e., subjected to "back-mutations" (see, e.g., morrison et al (1984) Proc. Natl. Acad. Sci.81 (21): 6851-6855;Neuberger et al (1984) Nature 312:604-608).

As used herein, "percent (%) sequence identity" of amino acid sequences, sequence identity "has art-recognized definitions that refer to the percentage of identity between two polypeptide sequences as determined by sequence alignment (e.g., by manual inspection or by a known algorithm). The determination may be made using methods known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, clustal Omega and FASTA software.

Herein, an amino acid sequence "derived from" or "derived from" a reference amino acid sequence is identical or homologous to part or all of the reference amino acid sequence. For example, an amino acid sequence derived from a heavy chain constant region of a human immunoglobulin may have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity to the wild-type sequence of the heavy chain constant region of the human immunoglobulin from which it is derived.

Non-critical regions in the polypeptide (e.g., non-critical amino acids of the CDR and framework regions of an antibody and the amino acids of the constant region) may be modified, e.g., substitutions, additions and/or deletions of one or more amino acids may be made, without altering the function of the polypeptide. Such polypeptides that are modified relative to a reference amino acid sequence (e.g., a single variable domain) may be referred to as "variants" of the reference amino acid sequence. Variants of a reference amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the sequence from which it is derived, or have substitutions, additions and/or deletions of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids compared to the sequence from which it is derived. Preferably, the substitution is a conservative substitution. Those skilled in the art will appreciate that amino acids in non-critical regions of a polypeptide may be substituted with suitable conserved amino acids and generally do not alter their biological activity (see, e.g., watson et al Molecular Biology of the Gene,4th Edition,1987,The Benjamin/Cummings pub. Co., p. 224). Suitable conservative substitutions are well known to those skilled in the art. Non-limiting examples of some common conservative substitutions of amino acid residues are set forth in the following table. In some cases, amino acid substitutions are non-conservative substitutions. It will be appreciated by those skilled in the art that amino acid mutations or modifications may be made to polypeptides to alter their properties, for example to alter the type of antibody glycosylation modification, to alter the ability to form interchain disulfide bonds, or to provide reactive groups for the preparation of antibody conjugates. Antibodies or antigen binding fragments thereof comprising such amino acid mutations or modifications are also encompassed within the scope of the antibodies or antigen binding fragments thereof of the invention.

"affinity" or "binding affinity" is used to measure the strength of the interaction between an antibody and an antigen through non-covalent interactions. The magnitude of the "affinity" can be reported as the equilibrium dissociation constant, K _D 。K _D It can be calculated by measuring the equilibrium association constant (ka) and equilibrium dissociation constant (kd): k (K) _D =kd/Ka. Affinity can be determined using conventional techniques known in the art, such as biofilm interference techniques (e.g., octet Fortebio detection systems can be employed), radioimmunoassay, surface plasmon resonance, enzyme-linked immunoassay (ELISA), or flow cytometry (FACS), among others.

In this context, an antibody and an antigen "specifically bind" refers to the binding of an antibody to an antigen with a relatively high affinity. Typically, K between a specifically bound antibody and antigen _D A value of at least about 10 ^-6 M to at least about 10 ^-9 M or less, e.g. at least about 10 ^-6 M, at least about 10 ^-7 M, at least about 10 ^-8 M, at least about 10 ^-9 M, at least about 10 ^-10 M or lower. In one embodiment, the anti-IL-17A antibodies or antigen-binding fragments thereof of the invention bind human IL-17A with high affinity, e.g., at 5X 10 ^-10 M or less, 1×10 ^-10 M or less, 5X 10 ^-11 M or less, 2X 10 ^-11 M or lower K _D Binds human IL-17A.

The anti-IL-17A antibodies or antigen-binding fragments, multispecific antibodies, or polynucleotides encoding the same of the invention may be isolated. As used herein, the expression "isolated" means that a substance (e.g., a polynucleotide or polypeptide) is isolated from the source or environment in which it is present, i.e., does not substantially comprise any other components.

In this context, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, which may generally include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

As used herein, the term "expression" refers to the production of RNA and/or polypeptides.

Herein, a "vector" is a vector used to introduce an exogenous polynucleotide into a host cell, which is amplified or expressed when the vector is transformed into an appropriate host cell. Vectors typically remain episomal, but may be designed to integrate a gene or portion thereof into the chromosome of the genome. As used herein, the definition of vector encompasses plasmids, linearized plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), and the like. Viral vectors include, but are not limited to, retroviral vectors (including lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, poxviral vectors, and baculovirus vectors, among others.

As used herein, an "expression vector" refers to a vector capable of expressing a polynucleotide of interest (including DNA and RNA). For example, in an expression vector, polynucleotide sequences encoding a polypeptide of interest (including DNA and RNA) may be operably linked to regulatory sequences capable of affecting the expression of the polynucleotide sequences (e.g., promoters and ribosome binding sites). Regulatory sequences may comprise promoter and terminator sequences, and optionally may comprise origins of replication, selectable markers, enhancers, polyadenylation signals, and the like. The expression vector may be a plasmid, phage vector, recombinant virus or other vector that, when introduced into an appropriate host cell, results in expression of the polynucleotide of interest. Suitable expression vectors are well known to those skilled in the art. The expression vector can be prepared by one skilled in the art as a vector that is replicable in a host cell, remains episomal in a host cell, or is integrated into the host cell genome, as desired.

As used herein, a "host cell" is a cell that is used to receive, hold, replicate, or amplify a vector. Host cells may also be used to express polypeptides encoded by polynucleotides or vectors. The host cell may be a eukaryotic cell or a prokaryotic cell. Prokaryotic cells such as E.coli (E.coli) or B.subtilis (Bacillus subtilis), fungal cells such as yeast cells or Aspergillus, insect cells such as S2 drosophila cells or Sf9, and animal cells such as fibroblasts, CHO cells, COS cells, heLa cells, NSO cells or HEK293 cells.

As used herein, the term "treatment" refers to an improvement in a disease/symptom, e.g., alleviation or disappearance of a disease/symptom, prevention or slowing of the occurrence, progression, and/or worsening of a disease/symptom. Thus, treatment includes prophylaxis, treatment and/or cure.

By "effective amount" is meant a dose sufficient to reduce the severity of the symptoms of the disease, increase the frequency and duration of the asymptomatic phase of the disease, or prevent injury or disability due to the affliction of the disease. An "effective amount" refers to that amount required to prevent, cure, ameliorate, block or partially block a disease or condition. The effective amount can be determined by one skilled in the art based on factors such as the age, physical condition, sex, severity of symptoms, particular composition or route of administration of the subject, and the like. An effective amount may be administered in one or more administrations.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, as is well known in the art (see, e.g., remington's Pharmaceutical sciences. Mediated by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995).

anti-IL-17A antibodies or antigen binding fragments thereof

In a general aspect, the present invention provides an anti-IL-17A antibody, or antigen-binding fragment thereof, comprising an immunoglobulin single variable domain that specifically binds IL-17A.

The term "IL-17A" as used herein refers to interleukin 17A. IL-17A is a pro-inflammatory cytokine, produced in a variety of T cells. IL-17A stimulates epithelial, endothelial and fibroblast cells to produce other pro-inflammatory cytokines and chemokines including, for example, IL-6, IL-8, G-CSF and MCP-1.IL-17A may be human IL-17A and non-human (e.g., camel, alpaca, mouse, rat, guinea pig, rabbit, goat, sheep, cow, horse, monkey, etc.) IL-17A. In one embodiment, the IL-17A is human IL-17A. The amino acid sequence of exemplary human IL-17A is shown in SEQ ID NO. 1.IL-17A may exist in vivo as IL-17A homodimers and IL-17A/F heterodimers (IL-17A/IL-17F).

In some embodiments, the anti-IL-17A antibodies or antigen-binding fragments thereof of the invention are capable of specifically binding IL-17A. In one embodiment, the anti-IL-17A antibody or antigen-binding fragment thereof is capable of neutralizing IL-17A. In some embodiments, the anti-IL-17A antibody or antigen-binding fragment thereof is capable of binding to IL-17A homodimers or IL-17A/F heterodimers. In some embodiments, the anti-IL-17A antibody or antigen-binding fragment thereof is capable of blocking IL-17A or IL-17A/F interaction with IL-17 RA.

In some embodiments, the anti-IL-17A antibodies or antigen-binding fragments thereof of the invention comprise a single variable domain of an immunoglobulin comprising CDR1, CDR2 and CDR3. In some embodiments, the single variable domain is a first family, second family, or third family single variable domain.

First family single variable domains

In some embodiments, the first family single variable domain comprises:

1) CDR1 comprising the amino acid sequence of SEQ ID NO: 68:

Xaa1-Xaa2-Xaa3-Xaa4-S-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9(SEQ ID NO:68)

2) CDR2 comprising the amino acid sequence of SEQ ID NO: 69:

Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-S-T-Xaa17(SEQ ID NO:69)

3) CDR3 comprising the amino acid sequence of SEQ ID No. 70:

D-Xaa18-Xaa19-Xaa20-Y-E-Xaa21-Xaa22-D-D-Y(SEQ ID NO:70)

In a preferred embodiment, the first family single variable domain comprises:

1) CDR1 comprising the amino acid sequence of SEQ ID NO 17, 38, 20, 23 or 35;

3) CDR3 comprising the amino acid sequence of SEQ ID NO. 19, 40, 22, 25 or 37.

In a specific embodiment, the first family single variable domain comprises:

Second family single variable domains

In some embodiments, the second family single variable domain comprises:

1) CDR1 comprising the amino acid sequence of SEQ ID NO: 71:

Xaa23-Xaa24-I-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-M-Xaa30(SEQ ID NO:71)

2) CDR2 comprising the amino acid sequence of SEQ ID NO: 72:

Xaa31-I-T-Xaa32-G-G-Xaa33-T-Xaa34(SEQ ID NO:72)

3) CDR3 comprising the amino acid sequence of SEQ ID NO. 28, 31, 34 or 43.

In a preferred embodiment, the second family single variable domain comprises:

1) CDR1 comprising the amino acid sequence of SEQ ID NO. 26, 29, 32 or 41;

2) CDR2 comprising the amino acid sequence of SEQ ID NO 27, 30, 33 or 42; and

3) CDR3 comprising the amino acid sequence of SEQ ID NO. 28, 31, 34 or 43.

In a specific embodiment, the second family single variable domain comprises:

Third family single variable domains

In some embodiments, the third family single variable domain comprises:

1) CDR1 comprising the amino acid sequence of SEQ ID NO: 73:

G-F-Xaa35-L-D-Xaa36-Xaa37-Xaa38-I-G(SEQ ID NO:73)

xaa35 is T or N; xaa36 is D or Y; xaa37 is D or Y; xaa38 is A or G;

2) CDR2 comprising the amino acid sequence of SEQ ID No. 74:

C-I-Xaa39-S-S-D-G-S-T-Y(SEQ ID NO:74)

xaa39 is S or T; and

3) CDR3 comprising the amino acid sequence of SEQ ID NO 46, 49 or 52.

In these embodiments, the single variable domain as described above has two pairs of disulfide bonds, one pair of disulfide bonds added inside the single variable domain that make it less protein aggregated than the disulfide bonds between one pair of CDR2 and CDR3 of a conventional single variable domain.

In a preferred embodiment, the third family single variable domain comprises:

1) CDR1 comprising the amino acid sequence of SEQ ID NO 44, 47 or 50;

2) CDR2 comprising the amino acid sequence of SEQ ID NO 45, 48 or 51; and

3) CDR3 comprising the amino acid sequence of SEQ ID NO 46, 49 or 52.

In a specific embodiment, the third family single variable domain comprises:

In some embodiments, the single variable domain as described above further comprises a framework region. The framework regions may each independently be derived from framework regions of immunoglobulins of any species.

In some embodiments, the framework regions are derived from immunoglobulins of alpaca. In one embodiment, the single variable domain comprises an amino acid sequence selected from the group consisting of: SEQ ID NO. 5, SEQ ID NO. 12, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16; wherein the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the sequence from which it is derived, or has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, additions, and/or deletions compared to the sequence from which it is derived. In one embodiment, the substitutions, additions and/or deletions do not occur in the CDR regions.

In some embodiments, the framework region is derived from a human immunoglobulin. In certain preferred embodiments, the single variable domain comprises a heavy chain framework region derived from a human immunoglobulin. Thus, in certain preferred embodiments, the anti-IL-17A antibodies or antigen-binding fragments thereof of the invention are humanized. The framework regions may comprise one or more non-human (e.g. camelid) amino acid residues, for example may comprise one or more amino acid back mutations in which the corresponding camelid amino acid residue is comprised. In one embodiment, the single variable domain comprises an amino acid sequence selected from the group consisting of: 65, 67 and 66; wherein the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the sequence from which it is derived, or has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, additions, and/or deletions compared to the sequence from which it is derived. In one embodiment, the substitutions, additions and/or deletions do not occur in the CDR regions.

In some embodiments, the anti-IL-17A antibodies or antigen-binding fragments thereof of the invention may further comprise an immunoglobulin constant region (e.g., a heavy chain constant region and/or a light chain constant region) portion linked to a single variable domain of the invention. Optionally, the constant region moiety is linked to a single variable domain of the invention by a linker.

Suitable immunoglobulin constant region (particularly heavy chain constant region portions of human immunoglobulins) portions may be selected and optionally modified to obtain antibodies having desired properties. For example, one or more cysteine residues may be introduced or removed (e.g., mutated by DNA recombination techniques) in the hinge region to promote or attenuate dimerization of the antibody; CH1, hinge region, CH2, and/or CH3 may be modified to extend or reduce serum half-life of the antibody, promote internalization or tissue penetration of the antibody, have improved or impaired binding to FcgammaRIIB (see, e.g., WO2008150494A 1), enhance or attenuate antibody-dependent cell-mediated cytotoxicity (ADADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), among others (see, e.g., caron, P.C., et al, J.exp. Med.176:1191-1195 (1992) and Shopes B.J.Immunol.148-2918 (1992)).

In some embodiments, the anti-IL-17A antibodies or antigen binding fragments thereof of the invention comprise a heavy chain constant region portion linked to a single variable domain of the invention. The heavy chain constant region portion may be derived from any immunoglobulin subtype or subclass, such as IgG, igM, igA, igD and IgE. Preferably, the heavy chain constant region portion is derived from human IgG, such as IgG1, igG2, igG3, and IgG4. The heavy chain constant region portion preferably comprises a hinge region, CH2, CH3, or a combination thereof. In a more preferred embodiment, the heavy chain constant region portion comprises an Fc fragment.

In some embodiments, the anti-IL-17A antibodies or antigen-binding fragments thereof of the invention further comprise an Fc fragment of an immunoglobulin. Preferably, the Fc fragment is an Fc fragment of human IgG 1. In one embodiment, the Fc fragment comprises the amino acid sequence of SEQ ID NO. 2.

In some embodiments, the anti-IL-17A antibodies or antigen binding fragments thereof of the invention comprise an immunoglobulin single variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO. 5, SEQ ID NO. 12, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, and SEQ ID NO. 16; and an Fc fragment of human IgG1 comprising the amino acid sequence of SEQ ID NO. 2. In a specific embodiment, the anti-IL-17A antibody or antigen-binding fragment thereof of the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 53-64.

In some embodiments, the anti-IL-17A antibodies or antigen-binding fragments thereof of the invention comprise an immunoglobulin single variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO. 65, SEQ ID NO. 67 and SEQ ID NO. 66; and an Fc fragment of human IgG1 comprising the amino acid sequence of SEQ ID NO. 2.

Furthermore, one skilled in the art is able to select any other polypeptide that can be linked to an anti-IL-17 antibody of the invention or antigen-binding fragment thereof (e.g., a single variable domain or heavy chain antibody) of the invention, as desired. Other polypeptides may be used to impart advantageous properties and/or reduce undesirable properties to the anti-IL-17A antibodies or antigen-binding fragments thereof of the invention, such as increasing the half-life, solubility, or absorption (e.g., facilitating penetration of the skin, phospholipid membranes, organelles, etc.) of the anti-IL-17A antibodies or antigen-binding fragments thereof; and/or reduce their immunogenicity, toxicity, or side effects. Antibodies or antigen-binding fragments comprising such polypeptides are also encompassed within the meaning of the anti-IL-17A antibodies or antigen-binding fragments thereof of the invention.

For example, serum albumin or a suitable fragment thereof may be linked to an anti-IL-17A antibody of the invention or an antigen-binding fragment thereof to increase its serum half-life (see, e.g., CN104884473A, WO2013177101A2, WO0027435A1 and WO0177137A 1). Other polypeptides may also be, for example, polypeptides for directing expression and secretion of antibodies or antigen-binding fragments thereof from a host cell, facilitating detection and/or isolation of antibodies or antigen-binding fragments, including but not limited to signal peptides (leader sequences), pro-lytic tags, affinity tags (e.g., polyhistidine tags (His) ₆ ) Or a glutathione S-transferase (GST) tag), a polypeptide comprising a protease cleavage site, and a reporter tag (e.g., a fluorescent protein). The other polypeptide may also be a biologically active polypeptide, such as a polypeptide or protein having therapeutic, binding or enzymatic activity. Non-limiting examples of biologically active polypeptides may include, but are not limited to: protein toxins (e.g., diphtheria toxin, ricin), enzymes (e.g., urease, horseradish peroxidase), and cytokines. The amino acid sequence of the polypeptides described above will be clear to a person skilled in the art.

The anti-IL-17A antibodies or antigen-binding fragments thereof of the invention are preferably linked directly to any polypeptide of interest (e.g., one or more of the polypeptides described above) or through a suitable linker. In a preferred embodiment, the anti-IL-17A antibody or antigen-binding fragment thereof is linked to one or more of the above polypeptides using a peptide linker, such that the anti-IL-17A antibody or antigen-binding fragment thereof of the invention can be expressed as a recombinant (fusion) protein.

The peptide linker may comprise an amino acid sequence of any length, in particular an amino acid sequence of 1 to 50, preferably 1 to 30, for example 1 to 10 amino acid residues. Exemplary peptide linkers may include, but are not limited to, poly glycine (G), poly alanine (a), poly serine (S), or combinations thereof, such as GGAS, GGGS, GGGSG or (G) ₄ S) _n Wherein n is an integer from 1 to 30, preferably from 1 to 10. The peptide linker may also be a hinge region or a functional equivalent thereof. Other suitable linkers may be organic compounds or polymers generally suitable for use in pharmaceutical proteins, including but not limited to polyethylene glycol.

The anti-IL-17A antibodies or antigen-binding fragment immunoglobulins of the present invention may be a single domain antibody, a heavy chain antibody, a humanized antibody or a chimeric antibody.

In some embodiments, the anti-IL-17A antibody or antigen-binding fragment thereof is capable of

1) Specifically binds IL-17A;

2) Neutralizing IL-17A;

3) Specifically binds to IL-17A homodimers or IL-17A/F heterodimers; and/or

4) Blocking the interaction of IL-17A or IL-17A/F with IL-17 RA.

Multispecific antibodies

In one aspect, the invention provides a multispecific antibody comprising a first antigen-binding portion which binds IL-17A and a second antigen-binding portion which binds a second antigen, wherein the first antigen-binding portion comprises an anti-IL-17A antibody or antigen-binding fragment thereof of the invention.

As used herein, the term "multispecific antibody" refers to an antibody that is capable of specifically binding to two or more (e.g., 2, 3, 4, 5, or 6) different epitopes. The multispecific antibody may be, for example, a bispecific, trispecific or tetraspecific antibody, which is capable of specifically binding 2, 3 or 4 epitopes, respectively. As used herein, the term "epitope" or "antigenic determinant" refers to a region of an antigen that specifically binds to an antigen binding site of an antibody. The second antigen may be an antigen other than IL-17A. The second antigen may also be IL-17A, which binds to a different epitope on IL-17A than an anti-IL-17A antibody or antigen binding fragment thereof of the invention. The determination of whether the epitope bound by the two antibodies is identical can be made using methods conventional in the art, for example, by ELISA, flow cytometry, or surface plasmon resonance to determine the competitive binding of the two antibodies to the same epitope.

The multispecific antibody may be a multivalent (e.g., 2, 3, 4 valent) antibody, i.e., it has multiple antigen binding sites.

Methods for constructing multispecific antibodies using an antibody or antigen-binding fragment of interest are well known to those skilled in the art (see, e.g., WO 93/08829;Suresh et al, (1986) Methods in Enzymology,121:210; and Traunecker et al, (1991) EMBO, 10:3655-3659). Multispecific antibodies can be generated and isolated using a variety of techniques known in the art.

As used herein, "first antigen binding portion" and "second antigen binding portion" refer to an amino acid sequence comprising an antigen binding site that is capable of binding to an epitope of an antigen, which definition falls within the meaning of an antibody or antigen binding fragment.

The first antigen binding portion may be any form of antibody or antigen binding fragment, including but not limited to single variable domains and heavy chain antibodies. In one embodiment, the first antigen binding portion comprises a single variable domain of the invention.

The second antigen binding portion may be an antibody or antigen binding fragment that binds any antigen of interest. In one embodiment, the second antigen is an antigen other than IL-17A. Antigens to which the second antigen binding portion may specifically bind may include pro-inflammatory cytokines and chemokines. As used herein, the term "pro-inflammatory cytokine" refers to a class of cytokines secreted by immune cells or other types of cells that promote inflammation.

The first antigen binding portion and the second antigen binding portion may optionally be connected by a linker (e.g., as described herein).

Nucleic acids, vectors and host cells

In another aspect, the invention provides a polynucleotide comprising a polypeptide encoding an anti-IL-17A antibody or antigen-binding fragment thereof of the invention.

The polynucleotides of the invention may be obtained using methods known in the art. For example, polynucleotides of the invention may be isolated from phage display libraries, yeast display libraries, immune animals (e.g., alpaca, mice, humanized mice, monkeys), immortalized cells (e.g., mouse B cell hybridoma cells, EBV-mediated immortalized B cells), or chemical synthesis. The polynucleotides of the invention may be codon optimized for the host cell used for expression.

In yet another aspect, the invention also provides a vector comprising a polynucleotide of the invention. In some embodiments, polynucleotides of the invention are cloned into an expression vector. The expression vector may further comprise additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes. The expression vector may also comprise a polynucleotide sequence encoding an additional polypeptide.

Polynucleotides of the invention may be present in one or more expression vectors.

The invention also provides a host cell comprising a polynucleotide or expression vector of the invention. The polynucleotides or expression vectors of the invention may be introduced into a suitable host cell using a variety of methods known in the art. Such methods include, but are not limited to, liposome transfection, electroporation, viral transduction, and calcium phosphate transfection, among others. In a preferred embodiment, the host cell is used to express an anti-IL-17A antibody or antigen-binding fragment thereof of the invention. Examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria, e.g., E.coli) and eukaryotic cells (e.g., yeast, insect cells, mammalian cells). Mammalian host cells suitable for antibody expression include, but are not limited to, myeloma cells, heLa cells, HEK cells (e.g., HEK 293 cells), chinese Hamster Ovary (CHO) cells, and other mammalian cells suitable for expression of antibodies.

The invention also provides a method of making an anti-IL-17A antibody or antigen-binding fragment thereof of the invention, comprising culturing a host cell of the invention under suitable conditions to express an anti-IL-17A antibody or antigen-binding fragment thereof of the invention, and isolating the antibody or antigen-binding fragment thereof from the host cell or culture thereof.

Antibody conjugates

The anti-IL-17A antibodies or antigen-binding fragments thereof of the invention may be conjugated to at least one detectable label. Conjugates comprising an anti-IL-17A antibody or antigen-binding fragment thereof of the invention and a detectable label are also encompassed within the meaning of an anti-IL-17A antibody or antigen-binding fragment thereof of the invention. Thus, in a further aspect, the invention also provides an antibody conjugate comprising an anti-IL-17A antibody or antigen-binding fragment thereof of the invention conjugated to at least one detectable label.

As used herein, "conjugated" refers to the attachment of two or more moieties to each other by covalent or non-covalent interactions. Preferably, the anti-IL-17A antibodies or antigen-binding fragments thereof of the invention are covalently conjugated to a detectable label.

The detectable label may be any label for detection including, but not limited to, a radioisotope (e.g. ²¹² Bi、 ²¹³ Bi、 ¹³¹ I、 ¹²⁵ I、 ¹¹¹ In、 ¹⁷⁷ Lu、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ¹⁵³ Sm (Sm) ⁹⁰ Y, etc.), biotin, colloidal gold, chemiluminescent labels, bioluminescent labels, and fluorophores (e.g., FITC, alexa Fluor 488, alexa Fluor 568, alexa Fluor 555, alexa Fluor 594, alexa Fluor 647, cy3, texas Red, cy5, and rhodoamine, etc.). In some embodiments, the detectable label is selected from the group consisting of a radioisotope, biotin, colloidal gold, a chemiluminescent label, a bioluminescent label, and a fluorescent group.

The detectable label may be conjugated directly or indirectly (e.g., via a linker) to an anti-IL-17A antibody or antigen-binding fragment thereof of the invention. The linker may contain reactive groups for covalent conjugation, such as amine, hydroxylamine, maleimide, carboxyl, phenyl, thiol, sulfhydryl or hydroxyl groups. In one embodiment, the linker is a chemical bond. In one embodiment, the linker comprises an amino acid or a peptide consisting of 2-10 amino acids.

Pharmaceutical composition

The invention also provides pharmaceutical compositions comprising an anti-IL-17A antibody or antigen-binding fragment thereof of the invention, and a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers can include, but are not limited to: diluents, binders and adhesives, lubricants, disintegrants, preservatives, vehicles, dispersants, glidants, sweeteners, coatings, excipients, preservatives, antioxidants (such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, ascorbyl palmitate, butylated Hydroxyanisole (BHA), butylated Hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like), solubilizing agents, gelling agents, softeners, solvents (such as water, alcohols, acetic acid, and syrups), buffers (such as phosphate buffers, histidine buffers, and acetate buffers), surfactants (such as nonionic surfactants, such as polysorbate 80, polysorbate 20, poloxamer, or polyethylene glycol), antibacterial agents, antifungal agents, isotonic agents (such as trehalose, sucrose, mannitol, sorbitol, lactose, glucose), absorption retarders, chelating agents, and emulsifiers. For liquid compositions, the carrier may be selected from one or more of the following: sterile diluents, for example water, saline solutions, preferably physiological saline, ringer's solution, isotonic sodium chloride, non-exertive oils such as synthetic mono-or diglycerides, polyethylene glycol, glycerol or other solvents; antimicrobial agents, such as benzyl alcohol or methylparaben; and agents for modulating tonicity such as sodium chloride or dextrose.

The pharmaceutical compositions provided herein may be in a variety of dosage forms including, but not limited to, solid, semi-solid, liquid (e.g., solution, emulsion, or suspension), powder, lotion, spray, patch, plaster, ointment, gel, cream, spray, paste, bioadhesive, or lyophilized formulation (e.g., for use after reconstitution). The pharmaceutical compositions of the invention may also be formulated as microemulsions, liposomes and micelles (see, e.g., CN107108730a and WO2016113557 A1). Preferably, the pharmaceutical composition may be formulated into dosage forms suitable for the desired route of administration, in particular topical, dermal, parenteral (e.g. intravenous, intramuscular, intrasternal, subcutaneous, e.g. by injection or infusion) and intranasal administration.

In embodiments involving transdermal administration, the pharmaceutical composition may comprise a skin penetration enhancer that aids in or enhances skin penetration. Skin penetration enhancers known in the art may be used, including but not limited to those described in CN107108730A, for example. The drug may also be administered using, for example, sonication, sonophoresis, electroporation, or using microneedle technology.

The pharmaceutical compositions may be prepared using methods well known in the pharmaceutical arts. For example, compositions intended for administration by injection may be prepared by combining a binding molecule of the invention with water to form a solution. Surfactants may be added to promote the formation of a homogeneous solution or suspension. The pharmaceutical composition may take the form of one or more dosage units. The pharmaceutical compositions may be prepared in ampoules, disposable syringes or multi-dose vials made of glass, plastic or other materials.

Detection of

The anti-IL-17A antibodies or antigen-binding fragments thereof or antibody conjugates of the invention can be used to detect the presence of IL-17A or its expression level. For example, the anti-IL-17A antibodies or antigen-binding fragments thereof or antibody conjugates provided herein can be used in situ, in vivo, ex vivo, and in vitro detection or imaging assays.

In some embodiments, the expression level of IL-17A in a sample and/or tracking cells expressing IL-17A is detected by using an antibody or antigen binding fragment or antibody conjugate of the invention.

In one aspect, the invention provides a method of detecting the presence of or determining the expression level of IL-17A in a sample comprising:

(a) Contacting an anti-IL-17A antibody of the invention or an antigen-binding fragment thereof (e.g., an antibody conjugate of the invention) with a sample; and

(b) Detecting the formation of an immune complex between the antibody or antigen binding fragment thereof (e.g., an antibody conjugate of the invention) and IL-17A in the sample or determining the amount of the immune complex, thereby detecting the presence of IL-17A in the sample or determining the level of expression of IL-17A in the sample.

The sample may be in any form, such as cells, tissues and body fluids.

The above assays may be performed using techniques known in the art, including but not limited to western immunoblotting, flow cytometry, radioimmunoassay (RIA), immunohistochemical assay (IHC), and enzyme-linked immunoassay (ELISA).

Treatment of

The present invention provides the use of an anti-IL-17A antibody or antigen-binding fragment thereof in the manufacture of a medicament for the treatment of IL-17A mediated diseases. The invention also relates to a method of treating an IL-17A mediated disease (e.g., an autoimmune disease) comprising administering to a subject in need thereof an anti-IL-17A antibody or antigen-binding fragment thereof of the invention or a pharmaceutical composition of the invention.

In some embodiments, IL-17A mediated diseases may include, for example, autoimmune diseases, inflammatory conditions, allergies and allergic conditions, hypersensitivity reactions, severe infections, and organ or tissue transplant rejection.

The anti-IL-17A antibodies or antigen-binding fragments thereof of the invention may be administered in combination with one or more anti-inflammatory drugs/immunosuppressants for the treatment of the above-described diseases. For a description of anti-inflammatory/immunosuppressive drugs see for example CN104884473a and WO2013177101A2.

The anti-IL-17A antibodies or antigen-binding fragments thereof or pharmaceutical compositions of the invention may be administered with other drugs at the same time or at different times, e.g., simultaneously, separately or sequentially.

In certain embodiments, the anti-IL-17A antibodies or antigen-binding fragments thereof or pharmaceutical compositions of the invention are administered topically to the skin of a subject in need thereof. Preferably, a pharmaceutical composition is used which is capable of penetrating at least the outer layer of the skin and which can thus be delivered skin or transdermally. For example, the anti-IL-17A or antigen-binding fragment thereof or pharmaceutical composition of the invention may be administered directly to diseased or healthy skin in the form of lotions, sprays, solutions, gels, ointments, pastes, plasters, patches, bioadhesives, suspensions, liposomes, micelles, microspheres and microemulsions (see e.g. CN107108730a and WO2016113557A 1) and the like.

The dosage of the anti-IL-17A antibodies or antigen-binding fragments thereof or pharmaceutical compositions of the invention will vary depending upon the particular formulation, mode of application, and the particular location, host and disease being treated. Other factors such as age, weight, sex, medical history, diet, time of administration, clearance, response sensitivity and severity of the disease should be considered. The precise dosage employed in the composition will also depend on the route of administration and the severity of the disease or condition, and should be determined at the discretion of the practitioner and the circumstances of each patient. The administration may be performed continuously or periodically within the maximum tolerated dose.

Kit for detecting a substance in a sample

The invention also provides kits comprising an anti-IL-17A antibody or antigen-binding fragment, antibody conjugate, multispecific antibody or pharmaceutical composition of the invention, and instructions for use. The kit may also comprise a suitable container. In certain embodiments, the kit further comprises a device for administering the drug. Typically, the kit further comprises a label for indicating the intended use and/or method of use of the kit contents. The term "label" includes any written or recorded material provided on or with or otherwise with the kit.

Advantageous effects

The anti-IL-17A antibodies or antigen-binding fragments thereof of the invention may achieve at least one of the following beneficial effects:

1) Specifically binds IL-17A with high affinity;

2) Is capable of specifically neutralizing IL-17A; and

3) Blocking the interaction of IL-17A with IL-17 RA.

In other aspects, the anti-IL-17A antibodies or antigen-binding fragments thereof (particularly nanobodies and heavy chain antibodies) of the invention have significant advantages over conventional antibodies, such as small molecular weight, high permeability, etc., such that they are capable of recognizing cryptic epitopes that are not accessible to conventional antibodies, are easy to produce, and are suitable for assembly with other antibodies into multispecific and multivalent antibodies.

Examples

The following examples are intended to be illustrative of the invention only and should not be construed as limiting the invention in any way. The experimental procedures, which are not specified in the following examples, were carried out according to conventional methods and conditions, or according to the commercial specifications.

Example 1 preparation of raw materials

1.1 preparation of recombinant antigen IL-17A-Fc

The DNA fragment encoding human IL-17A (NCBI Gene ID:3605, SEQ ID NO: 1) was synthesized by general Biotechnology Co., ltd, and was ligated to the 5' -end of the DNA fragment encoding human IgG1 Fc (SEQ ID NO: 2) by PCR to obtain a nucleic acid encoding the recombinant antigen IL-17A-Fc. The nucleic acid is then cloned into eukaryotic expression vector pcDNA3.4-TOPO (Invitrogen) by homologous recombination to obtain the expression vector for recombinant antigen IL-17A-Fc. The expression vector of recombinant antigen IL-17A-Fc was transformed into E.coli DH 5. Alpha. And plasmids were extracted for eukaryotic expression.

Recombinant antigen IL-17A-Fc was expressed by the Expi293 transient expression system (ThermoFisher, A14635). After 7 days of transfection, the cell expression supernatant was centrifuged at 15000g for 10min at high speed, and the obtained Fc tagged protein expression supernatant was affinity purified with MabSelect SuRe LX (GE, 17547403), and then eluted with 100mM sodium acetate (pH 3.0), followed by neutralization with 1M Tris-HCl; the eluted protein was subjected to ultrafiltration and concentration tube (Millipore, UFC 901096) exchange to PBS buffer, and frozen at-80℃for use.

1.2 preparation of positive control antibodies

The positive control antibody used in the present application was anti-IL-17A antibody Studimumab, and plasmids containing the Studimumab heavy chain (SEQ ID NO: 3) gene and the Studimumab light chain (SEQ ID NO: 4) gene were constructed by molecular cloning methods, respectively, according to the sequence synthesis disclosed in patent application US 20170355762A. The remaining steps are described in example 1.1.

EXAMPLE 2 recombinant antigen IL-17A-His alpaca immunization and serum titre detection

2.1 immunization of animals

Immunization was performed by subcutaneous injection, and 2 alpacas NSY007 and NSY008 (Nanchang Dajia technologies Co., ltd.) were immunized with the recombinant antigen IL-17A-His (Novoprotein Co., ltd., cat. C774). The single immunization dose was 500 μg, supplemented with CFA/IFA (Freund's complete adjuvant and Freund's incomplete adjuvant), 1 time every 2 weeks, 4 total times.

2.2 serum potency detection

And after the 2 nd, 3 rd and 4 th immunization are finished, blood is taken to detect the titer of the targeted recombinant antigen IL-17A-His antibody in serum. The specific detection method comprises the following steps: the recombinant antigen IL-17A-His was diluted with PBS to a final concentration of 2. Mu.g/mL, 30. Mu.L of the dilution was added to ELISA plates and coated overnight at 4 ℃. PBST was rinsed three times on the same day for immunotiter determination, then blocked with PBST containing 5% skimmed milk for two hours at room temperature, and then rinsed three times with PBST. The non-immunized negative and post-immunized sera were diluted with PBS on another dilution plate, 2000-fold diluted in the first well, and then 2-fold gradient diluted in the next 7 wells. The diluted serum was added to the first ELISA plate, incubated at room temperature for 1h, and after three times of PBST plate washing, secondary Anti-antibody IgG-HRP (Millipore, MAC 129) was added at 1:7000, and incubated at room temperature for 0.5h. After incubation, the plates were washed six times with PBST, developed with TMB (SurModics, TMBS-1000-01), and the reaction was stopped by adding 2M HCl based on the developed results, and the plates were read by a microplate reader (Molecular Devices, specterMax 190) at OD 450.

The results are shown in Table 1, and the titers of antibodies targeting the recombinant antigen IL-17-His in two alpaca sera reached over 1:256K after the 4 immunizations were completed.

TABLE 1IL-17A immune alpaca serum IgG potency assay

Example 3 phage display library construction and targeting IL-17A nanobody screening

Cloning antibody genes of alpaca peripheral blood B cells immunized with recombinant antigen IL-17A-His as described in example 2, constructing a nanobody gene phage display library, and screening the library with recombinant antigen IL-17A-Fc (as described in example 1.1) as screening antigen to obtain a plurality of nanobodies specifically binding IL-17A protein.

3.1 construction of phage display library of camel nanobodies

Peripheral mononuclear cells (Peripheral Blood Mononuclear Cell, PBMC) were isolated from Ficoll-Paque density gradient (GE, cat. No.: 17144003S), total RNA was extracted from the isolated PBMC cells and reverse transcribed into cDNA. Degenerate primers were designed based on the case of VHH antibody germ line gene (germline) and the DNA fragment encoding VHH-CH2 was obtained after PCR amplification and recovery of the PCR product by agarose gel electrophoresis. Then, a DNA fragment encoding a variable domain (VHH) was amplified by a secondary PCR method using the DNA fragment encoding VHH-CH2 as a template (Sabir JS et al, C R biol. (2014) 337 (4): 244-249). The DNA fragment encoding the VHH is then digested and purified and then constructed into a phage display vector. Finally, the VHH-expressing vector was transformed into competent E.coli SS320 (Lucigen, MC 1061F) by electrotransfer (Bio-Rad, microPulser) and the transformed E.coli SS320 bacterial liquid was plated on ampicillin-resistant 2-YT solid plates. Library capacity of 10 was determined by gradient dilution plating ⁹ A stage. Adding bacterial liquid of the nanobody gene library into fresh 2-YT liquid culture medium, placing in a shaking table at 37 ℃ and 220rpm for culturing until logarithmic growth phase, adding VSCM13 helper phage (purchased from Stratagene) in an amount of 50 times of the bacterial number (namely, the multiplicity of infection (MOI) is about 50), and finally obtaining the phage display library of camel-origin nanobody.

3.2 screening of antibody Gene phage display libraries

3.2.1 screening of antibody Gene phage display library by magnetic bead method

The magnetic bead screening is based on labeling recombinant antigen IL-17A-Fc with biotin, then combining with streptavidin-coupled magnetic beads, and performing incubation, washing and elution of antigen-coupled magnetic beads and antibody gene phage display library, so that monoclonal antibodies specific to the antigen can be enriched in large quantities.

The specific method comprises the following steps: first, the biotin-labeled IL-17A-Fc was incubated with streptavidin-coupled magnetic beadsThe biotin-labeled IL-17A-Fc was allowed to bind to the magnetic beads. The IL-17A-Fc binding beads and the phage library were incubated for 2h at room temperature. After washing 6-8 times with PBST, nonspecifically adsorbed phage was removed, trypsin (Gibco, 25200072) was added, gently mixed and reacted for 20min to elute specifically bound antibody-displaying phage. Subsequently, the log-phase E.coli SS320 was infected with the eluted phage and allowed to stand for 30min, followed by cultivation at 220rpm for 1 hour; adding VSCM13 helper phage, standing for 30min, and culturing at 220rpm for 1 hr; centrifuging and changing the liquid to C ⁺ /K ⁺ In 2-YT medium, the resulting phage was used for the next round of panning.

3.2.2 screening of antibody Gene phage display library by Immunotube method

The principle of the immune tube screening is to coat IL-17A protein on the surface of an immune tube with high adsorption capacity, and finally enrich specific monoclonal antibodies aiming at antigens through the panning process of adding phage display antibody libraries into the immune tube and incubating, washing and eluting with antigen proteins adsorbed on the surface of the immune tube.

The specific method comprises the following steps: in the first round of screening, 1mL of IL-17A-Fc (30. Mu.g/mL) was added to the immune tube and coated overnight at 4 ℃; the coating liquid is discarded the next day, and PBS (phosphate buffered saline) added with 5% milk is added for sealing for 2 hours; after PBS is rinsed twice, phage library containing anti-IL-17A nano antibody display is added, and incubation is carried out for 2 hours; rinsing to remove non-specifically bound phage, then adding 0.8ml of 0.05% edta pancreatin digest to the immune tube for eluting phage that specifically bind to the antigen of interest; then, infecting the colibacillus SS320 in the logarithmic phase by using the eluted phage, standing for 30min at 37 ℃, then culturing for 1h under the condition of 220rpm, adding the VSCM13 helper phage, standing for 30min, and continuing culturing for 1h under the condition of 220 rpm; centrifuging and changing the liquid to C ⁺ /K ⁺ In 2-YT medium, and continued to culture at 30℃and 220rpm overnight. The following day phages were precipitated for the next round of screening.

3.3 selection of monoclonal

ELISA detection is carried out on phage pools obtained by magnetic bead method screening and phage pools obtained by immune tube method screening of each round of elution to evaluate the enrichment effect. For the third round of enrichment, a large number of monoclonal were picked for ELISA primary screening (method reference example 3 in CN112745391 a). 134 nanobodies which are in sequence diversity and combined with IL-17A antigen are obtained through sequencing analysis and ELISA combined with a primary screen, 30 nanobodies are selected for ELISA affinity and blocking re-detection after blocking the primary screen (the method is described in example 3 in CN 112745391A), and the results are shown in figures 1A-1C and 2A-2C. Finally, 12 monoclonal antibodies are selected according to the affinity and blocking activities. The complementarity determining region sequences of the anti-IL-17A nanobody were determined by naming the nanobody with clone numbers and defining the CDRs by AbM, as shown in Table 2.

TABLE 2 variable region sequences of anti-IL-17A nanobodies

EXAMPLE 4 construction, expression and purification of heavy chain antibodies

The 5 nanobodies obtained in example 3 were constructed as human IgG1 subtype, forming heavy chain antibodies of VHH-Fc, with specific amino acid sequences as shown in table 3.

4.1 plasmid construction

From the candidate-containing monoclonal strains obtained by screening, a DNA fragment encoding VHH was obtained by PCR amplification. The DNA fragments encoding each VHH were constructed on a modified eukaryotic expression vector plasmid pcDNA3.4-TOPO (Invitrogen) containing a human IgG1 Fc fragment (SEQ ID NO: 2) by homologous recombination to obtain recombinant plasmids containing the full-length gene of the complete VHH-Fc (heavy chain antibody). The recombinant plasmid was transformed into E.coli DH 5. Alpha. And the plasmid was extracted for expression in eukaryotic cell systems.

4.2 expression and purification of antibodies

The heavy chain antibody was expressed by the expcho transient expression system (Thermo Fisher, a 29133) as follows: on the day of transfection, cell density was confirmed to be 7×10 ⁶ Up to 1X 10 ⁷ Cell viability about living cells/mL>98%, at this time, preheated at 37 ℃CFresh ExpiCHO expression medium adjusted to 6X 10 cells final concentration ⁶ Individual cells/mL. OptiPRO pre-cooled at 4deg.C ^TM SFM dilution of plasmid of interest (1. Mu.g plasmid was added to 1mL of the medium), while using OptiPRO ^TM SFM dilution of Expifectamine ^TM CHO, mixing the two materials in equal volume, and gently stirring to obtain the product ^TM The CHO/plasmid DNA mixture was incubated at room temperature for 1-5min, slowly added to the prepared cell suspension while gently shaking, and finally placed in a cell culture shaker at 37℃and 8% CO ₂ Culturing under the condition.

18-22h after transfection, expiCHO was added to the culture broth ^TM Enhance and ExpiCHO ^TM Feed, shake flask placed on a shaker at 32℃and 5% CO ₂ Culturing was continued under the conditions. On day 5 post transfection, the same volume of ExpiCHO was added ^TM Feed, slowly add while gently mix the cell suspension. After 7-15 days of transfection, the cell culture supernatant expressing the protein of interest was centrifuged at 15000g for 10min at high speed, the resulting supernatant was affinity purified with MabSelect SuRe LX (GE, 17547403), the protein of interest was eluted with 100mM sodium acetate (pH 3.0), then neutralized with 1M Tris-HCl, and finally the resulting protein was changed to PBS buffer by ultrafiltration of a concentrate tube (Millipore, UFC 901096).

TABLE 3 amino acid sequences of anti-IL-17 heavy chain antibodies

EXAMPLE 5 physicochemical Property identification of heavy chain antibody

5.1 SDS-PAGE identification of heavy chain antibodies

Non-reducing (non-denaturing) SDS-PAGE sample preparation: 1. Mu.g of heavy chain antibody or quality control IPI (i.e., ipilimumab (Ipilimumab), prepared by the method of example 4) was added to a 5 XSDS loading buffer (containing iodoacetamide at a final concentration of 40 mM), heated in a dry bath at 75℃for 10min, cooled to room temperature, and centrifuged at 12000rpm for 5min to obtain the supernatant. Reduction (denaturation) SDS-PAGE sample preparation: 2. Mu.g of heavy chain antibody or quality control IPI was added to 5 XSDS loading buffer (containing 5mM final concentration of DTT), heated in a dry bath at 100℃for 10min, cooled to room temperature, and centrifuged at 12000rpm for 5min to obtain the supernatant. The supernatant was gel-electrophoresed with Bis-tris 4-15% gradient gel (gold srey) and the protein bands were visualized by coomassie brilliant blue staining. Protein gels with chromogenic protein bands were scanned using an EPSON V550 color scanner (decolorized to gel background transparent) and reduced and non-reduced band purities were calculated by ImageJ according to peak area normalization.

The results are shown in FIGS. 3A-3B and Table 4: the apparent relative molecular weight of the bands of the heavy chain antibody and the quality control IPI non-reducing rubber is about 80kD and about 150kD respectively, the apparent relative molecular weight of the bands of the heavy chain antibody reducing rubber is about 40kD, and the quality control IPI is about 55kD and about 25kD respectively. The heavy chain antibodies analyzed by reduced and non-reduced SDS-PAGE were of molecular weight consistent with the expected size and purity greater than 90%.

5.2 SEC-HPLC monomer purity identification of heavy chain antibodies

Material preparation: 1. mobile phase: 150mmol/L phosphate buffer, pH 7.4; 2. sample preparation: the heavy chain antibody and the quality control IPI are diluted to 0.5mg/mL by mobile phase solution. Agilent HPLC 1100 column (XBIridge BEH SEC 3.5 μm,7.8mm I.D. times.30 cm, waters) flow rate was set at 0.8mL/min, sample volume 20. Mu.L, VWD detector wavelengths 280nm and 214nm.

SEC-HPLC analysis of heavy chain antibodies gave the following results: the percentage of high molecular aggregates, antibody monomers and low molecular aggregates in the samples were calculated according to the area normalization method and the results are shown in table 4, where the monomer purity of all heavy chain antibodies was greater than 96.0%.

TABLE 4 physicochemical data of heavy chain antibodies

Example 6 determination of affinity and blocking Activity of heavy chain antibodies based on ELISA method

ELISA-based methods demonstrated the affinity of heavy chain antibodies for human IL-17A antigen, and ELISA-based methods demonstrated the effect of heavy chain antibodies blocking the binding of IL-17A and IL-17 RA.

6.1 detection of affinity Activity of heavy chain antibodies for human IL-17A based on ELISA

96-well plates were coated with IL-17A-Fc (2. Mu.g/mL, 30. Mu.L/well) overnight at 4 ℃; the next day, 96-well plates were washed 3 times with PBST and blocked with 5% skimmed milk for 2h; after washing the plates 3 times with PBST, gradient diluted antibodies to be tested (heavy chain antibodies VHH1, VHH2, VHH3, VHH8 and VHH9; ipilimumab (IPI) as negative control; secukinumab as positive control) or PBS only (blank control) were added and incubated for 1h; after 3 washes with PBST, secondary antibodies were added: for heavy chain antibodies, anti-VHH-HRP (Kirschner Biotechnology, A01861); for secukinumab and ipilimumab, anti-kappa-HRP (Millipore, a 18853) and incubated for 1h; after incubation, PBST plates were washed six times, and developed with TMB (SurModics, TMBS-1000-01); based on the color development, the reaction was quenched by addition of 2M HCl and the plate was read by a microplate reader (Molecular Devices, specterMax 190) at OD 450.

The results are shown in FIGS. 4A-4C, which demonstrate that all heavy chain antibodies have high affinity activity for the antigen human IL-17A. EC due to the difference between heavy chain antibodies and control antibodies using secondary antibodies ₅₀ There is no comparability.

6.2 detection of the Effect of heavy chain antibodies on blocking IL-17A and IL-17RA binding based on ELISA

96-well plates were coated with 4. Mu.g/mL, 30. Mu.L/well of human IL-17RA-His protein (Sino Biological, cat# 10895-H08H) overnight at 4 ℃; the next day, 96-well plates were washed 3 times with PBST and blocked with 5% skimmed milk for 2h; then premixing heavy chain antibody or positive control antibody Stuzumab (PBS as blank control) with biotin-labeled IL-17A-Fc (1.5 mug/mL) for 0.5h in advance, adding the heavy chain antibody or positive control antibody Stuzumab into a 96-well plate after the sealing is completed and the plate washing is completed, and incubating for 1h; after 3 washes with PBST, neutravidin-HRP (Therofisther, 31001) was added and incubated for 1h; after incubation, PBST plates were washed six times, and developed with TMB (SurModics, TMBS-1000-01); based on the color development, the reaction was quenched by addition of 2M HCl and the plate was read by a microplate reader (Molecular Devices, specterMax 190) at OD 450. The results are shown in FIGS. 5A-5C. The results show that all heavy chainsThe antibodies have better effect of blocking the combination of IL-17A and IL-17 RA: the blocking effect of heavy chain antibodies VHH3, VHH4, VHH5, VHH6, VHH8, VHH10, VHH11, VHH12 and VHH13 was comparable to that of secukinumab; the blocking effect of the heavy chain antibodies VHH1, VHH2 and VHH9 is significantly better than that of the Stuzumab, wherein the IC of VHH1 ₅₀ IC of = 7.183nM, VHH2 ₅₀ IC of VHH 9= 7.387nM ₅₀ IC of secukinumab=5.766 nM ₅₀ ＝10.81nM。

Example 7 determination of affinity of heavy chain antibodies by biofilm layer interferometry

The binding affinity of heavy chain antibodies to the antigen human IL-17A was detected by biofilm layer interference techniques (using Fortebio equipment). As a comparison, the binding affinity of the control antibody, secukinumab, to the antigen human IL-17A was also determined.

IL-17A-His as antigen was diluted to 10. Mu.g/mL in 10 XKB buffer (10 XPBS containing 1% BSA, 0.5% Tween 20), and heavy chain antibody was diluted 2-fold in 10 XKB buffer, followed by dilution from 80nM to 1.25nM. Under dark conditions, a 10 XKB buffer prewetting sensor (Anti-Penta-HIS, HIS1K, fortebio, calif.) was used, and after at least 10min, the sample plate (Greinier Bio, PN 655209) was started and tested error free and then following the pre-set procedure. Firstly, combining an antigen and a sensor for 120s, after the combination is completed and balanced for 30s in 10 XKB buffer, transferring the sensor combined with the antigen into antibody diluents with different concentrations to combine for 120s, after signals are stabilized, transferring the sensor into the 10 XKB buffer, keeping dissociation time to be 120s, and finally obtaining K through combination dissociation data fitting of antibodies with different concentrations _D (affinity kinetic constant), K _on (binding constant) and K _off (dissociation constant), K _on Can be written as K _a ，K _off Can be written as K _d 。

The detection results are shown in Table 5. The results show that the heavy chain antibody binds to recombinant antigen IL-17A-His K _D At 4.57×10 ^-10 M-3.17×10 ^-11 M, the heavy chain antibodies were shown to have higher affinity for IL-17A. Among them, the heavy chain antibodies VHH1, VHH2, VHH3, VHH5, VHH6, VHH8, VHH9 and VHH11 all showed better affinity than secukinumab.

Table 5 table of antibody affinity assay data

Antibody name	K _D (M)	K _a (1/Ms)	K _d (1/s)
				Secukinumab	2.51E-10	5.31E+05	1.33E-04
VHH1	1.72E-10	8.62E+05	1.48E-04
				VHH2	1.53E-10	9.49E+05	1.46E-04
VHH3	1.01E-10	7.42E+05	7.48E-05
				VHH4	4.57E-10	8.36E+05	3.82E-04
VHH5	3.17E-11	1.56E+05	4.95E-06
				VHH6	2.09E-10	1.49E+06	3.13E-04
VHH8	1.22E-11	2.81E+05	3.42E-06
				VHH9	2.37E-11	2.63E+06	6.25E-05
VHH10	NA	1.37E+05	NA
				VHH11	1.35E-10	4.66E+05	6.28E-05
VHH12	NA	9.36E+04	NA
				VHH13	NA	3.41E+05	NA

Remarks: NA indicates that the antibody did not dissociate within 120s after binding, and therefore K was not calculated _d K is as follows _D Values.

EXAMPLE 8 determination of heavy chain antibody neutralization Activity

The cell biological activity of anti-IL-17A heavy chain antibodies was tested by detecting that anti-IL-17A heavy chain antibodies inhibited IL-17A from stimulating IL-6 secretion by HeLa cells. The specific method comprises the following steps:

HeLa cells (cell bank of the department of Chinese sciences) were resuscitated and cells which were passaged 2 to 4 times and had good growth state were used for experiments. Will be 1X 10 ⁵ HeLa cells were seeded at 100. Mu.L per well into new 96-well cell culture plates and incubated overnight in a 37℃cell incubator; the next day, heavy chain antibody to be tested and positive control antibody, studies, were diluted in DMEM medium, 100ng/mL IL-17A-His protein and 30ng/mL TNF-alpha protein (Sino Biological, 10602-H01H) were added, and the antibody and protein mixture was added to 96-well cell plates and incubated in a 37℃cell incubator for 24 hours. After the completion of the culture, the cell culture supernatant was harvested and subjected to detection and quantitative detection using an IL-6ELISA detection kit (BD, 555220).

The results of the assays are shown in FIGS. 6A-6K, which show that IL-17A and TNF-alpha induced IL-6 secretion is significantly inhibited in HeLa cells supplemented with heavy chain antibodies or Studies, indicating that both heavy chain antibodies have good IL-17A neutralizing activity: wherein, the neutralization activities of VHH1, VHH2, VHH3, VHH4, VHH5, VHH9, VHH11, VHH12 and VHH13 are all superior to that of the secukinumab; the neutralizing activity of VHH6 and VHH8 was comparable to that of secukinumab.

Example 9 nanobody humanization

In order to reduce the immunogenicity which may be caused by camel-derived nanobodies, the framework regions of the nanobodies are subjected to humanized mutation and back mutation to obtain heavy chain antibodies with higher degrees of humanization while maintaining the affinity of the humanized antibodies for the antigen.

9.1 nanometer antibody humanized transformation process

The heavy chain antibodies (corresponding VHH amino acid sequences of heavy chain antibodies VHH1, VHH2 and VHH 9) corresponding to clone numbers NB27A-15, 2-NB27B-19 and NB26B-12 are selected for humanization modification, the antibody sequences and a human antibody germ line gene database are compared, 1-3 germ line genes with higher homology with each VHH sequence are found, meanwhile, the drug property of the germ line genes is considered, a proper germ line gene template is selected for comparison, and the number of non-human sequence sites in a VHH framework region is analyzed. Performing homologous modeling on the VHH, wherein the homologous modeling refers to a nanobody model of the PDB database. Combining the simulated structural model of VHH and the non-human site situation, carrying out combined back mutation design (simultaneously avoiding introducing potential post-translational modification sites), and designing sequences with different degrees of humanization. The VHHs of the heavy chain antibodies VHH1, VHH2 and VHH9 were humanised to give the humanised antibodies VHH1-huVH4, VHH2-huVH3 and VHH9-huVH3, respectively, with the amino acid sequences shown in SEQ ID NOS 65, 66 and 67, respectively.

Expression vectors for humanized antibodies VHH1-huVH4, VHH2-huVH3 and VHH9-huVH3 were constructed as described in example 4.1. The humanized antibodies VHH1-huVH4, VHH2-huVH3 and VHH9-huVH3 comprise the VHH amino acid sequences (SEQ ID NOS: 65, 66 and 67) as described above, respectively, and the Fc fragment of human IgG1 (SEQ ID NO: 2). Expression and purification of humanized antibodies are described in example 4.2.

9.2 detection of affinity Activity of humanized antibodies (ELISA)

The affinity of the humanized antibodies VHH1-huVH4, VHH2-huVH3 and VHH9-huVH3 for antigen IL-17A was determined by ELISA and compared to the respective parent antibodies.

The specific method comprises the following steps: 96-well plates were coated with recombinant protein IL-17A-His (2. Mu.g/mL, 30. Mu.L/well) overnight at 4 ℃. The next day, 96-well plates were washed 3 times with PBST and blocked with 5% skimmed milk for 2h; after washing the plates 3 times with PBST, gradient diluted antibodies to be tested (humanized antibodies VHH1-huVH4, VHH2-huVH3 and VHH9-huVH3; parental heavy chain antibodies VHH1, VHH2 and VHH9; secukinumab as positive control; ipilimumab (IPI) as negative control) were added and incubated for 1h; after 3 washes with PBST, secondary anti-human Fc-HRP (Jackson Immuno Research, 109-035-008) was added and incubated for 1h; after incubation, PBST plates were washed six times, and developed with TMB (SurModics, TMBS-1000-01); based on the color development, the reaction was quenched by addition of 2M HCl and the plate was read by a microplate reader (Molecular Devices, specterMax 190) at OD 450.

The results are shown in figures 7A-7B, which demonstrate that humanized antibodies VHH1-huVH4, VHH2-huVH3 and VHH9-huVH3 have an affinity for antigen IL-17A comparable to the respective parent heavy chain antibodies VHH1, VHH2 and VHH9, and are all significantly better than the positive control antibody, secukinumab.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of details may be made to adapt to a particular situation and the invention is intended to be within the scope of the invention. The scope of the invention is given by the appended claims and any equivalents thereof.

Sequence listing

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Cys Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 5

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH1/NB27A-15 variable region

<400> 5

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val

35 40 45

Ala Thr Val Glu Ile Gly Gly Ser Ser Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Val Asp Trp Lys Trp Tyr Glu Val Asp Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210> 6

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH2/2-NB27B-19 variable region

<400> 6

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ser His

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Thr Arg Ser Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Thr Asp Tyr Arg Tyr Tyr Glu Ser Ile Asp Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210> 7

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH3/2-NB27A-93 variable region

<400> 7

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Ala Ser Ile Phe Ser Ile His

20 25 30

Ala Ile Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Ser Asn Val Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Tyr Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Met Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Trp Thr Tyr Tyr Glu Val Asp Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Gln Val Thr Val Ser Ser

115

<210> 8

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH4/NB27B-6 variable region

<400> 8

Glu Val Gln Val Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Ala Ser Ile Phe Asn Ala His

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Thr Ser Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Val Asp Arg Arg Tyr Tyr Asp Leu Ala Tyr Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210> 9

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH5/2-NB27A-95 variable region

<400> 9

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser Ile Phe Ser Ile His

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ser Ile Thr Tyr Gly Gly Asn Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Asp Asn Tyr Tyr Asp Glu Tyr Ala Pro Trp Gly Gln Gly Thr

100 105 110

Gln Val Thr Val Ser Ser

115

<210> 10

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH6/NB27A-33 variable region

<400> 10

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Gly Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Glu Tyr

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Leu Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Pro Glu Leu Phe Arg Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210> 11

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH8/2-NB27A-48 variable region

<400> 11

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Ala Ser Ile Phe Ser Ile His

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Thr Asn Ser Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Gly Arg Tyr Tyr Glu His Asp Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Gln Val Thr Val Ser Ser

115

<210> 12

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH9/NB26B-12 variable region

<400> 12

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Ile Ser Asp Phe

20 25 30

Pro Met Ser Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Ser Ala Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Val Asp Trp Lys Trp Tyr Glu Val Asp Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210> 13

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH10/2-NB27A-10 variable region

<400> 13

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Ile Ser Ile Tyr

20 25 30

Ser Met Asn Trp Tyr Arg Gln Ala Pro Gly Asn Glu Arg Glu Leu Val

35 40 45

Ala Arg Ile Thr Arg Gly Gly Thr Thr Asn Tyr Gly Asp Ser Val Lys

50 55 60

Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ala Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Gly Ala Asp Gly Gly Ile Trp Gly Gln Gly Thr Gln Val Thr Val

100 105 110

Ser Ser

<210> 14

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH11/2-NB27A-120 variable region

<400> 14

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Asp

20 25 30

Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Cys Ile Ser Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Thr Cys Gln Ile Tyr Gly Leu Gly His Asp Ala Ala Ala Pro Leu

100 105 110

Gly Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 15

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH12/2-NB27B-130 variable region

<400> 15

Glu Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Leu Asp Tyr Tyr

20 25 30

Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Ile

35 40 45

Ser Cys Ile Thr Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Gly Thr Val Asn

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asp Cys Met Gly Arg Glu Phe Gly Pro Ala Ser Ile Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 16

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH13/NB27B-22 variable region

<400> 16

Glu Val Arg Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr

20 25 30

Gly Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Cys Ile Ser Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Val Ala Glu Ile Tyr Leu Ser Gly Tyr Gly Cys Tyr Val Asp Gly

100 105 110

Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 17

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH1 / NB27A-15 CDR1

<400> 17

Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser

1 5 10

<210> 18

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH1 / NB27A-15 CDR2

<400> 18

Thr Val Glu Ile Gly Gly Ser Ser Thr Asn

1 5 10

<210> 19

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH1 / NB27A-15 CDR3

<400> 19

Asp Trp Lys Trp Tyr Glu Val Asp Asp Tyr

1 5 10

<210> 20

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH2 / 2-NB27B-19 CDR1

<400> 20

Gly Ser Ile Phe Ser Ser His Ala Met Gly

1 5 10

<210> 21

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH2 / 2-NB27B-19 CDR2

<400> 21

Ala Ile Thr Arg Ser Gly Ser Thr Asp

1 5

<210> 22

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH2 / 2-NB27B-19 CDR3

<400> 22

Asp Tyr Arg Tyr Tyr Glu Ser Ile Asp Asp Tyr

1 5 10

<210> 23

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH3 / 2-NB27A-93 CDR1

<400> 23

Ala Ser Ile Phe Ser Ile His Ala Ile Ala

1 5 10

<210> 24

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH3 / 2-NB27A-93 CDR2

<400> 24

Ala Ile Ser Asn Val Gly Ser Thr Asn

1 5

<210> 25

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH3 / 2-NB27A-93 CDR3

<400> 25

Asp Trp Thr Tyr Tyr Glu Val Asp Asp Tyr

1 5 10

<210> 26

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH4 / NB27B-6 CDR1

<400> 26

Ala Ser Ile Phe Asn Ala His Ala Met Gly

1 5 10

<210> 27

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH4 / NB27B-6 CDR2

<400> 27

Ala Ile Thr Ser Gly Gly Ser Thr Asp

1 5

<210> 28

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH4 / NB27B-6 CDR3

<400> 28

Asp Arg Arg Tyr Tyr Asp Leu Ala Tyr Asp Tyr

1 5 10

<210> 29

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH5 / 2-NB27A-95 CDR1

<400> 29

Gly Ser Ile Phe Ser Ile His Ala Met Gly

1 5 10

<210> 30

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH5 / 2-NB27A-95 CDR2

<400> 30

Ser Ile Thr Tyr Gly Gly Asn Thr Asn

1 5

<210> 31

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH5 / 2-NB27A-95 CDR3

<400> 31

Asp Asp Asn Tyr Tyr Asp Glu Tyr Ala Pro

1 5 10

<210> 32

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH6 / NB27A-33 CDR1

<400> 32

Gly Ser Ile Phe Ser Glu Tyr Ala Met Gly

1 5 10

<210> 33

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH6 / NB27A-33 CDR2

<400> 33

Thr Ile Thr Ser Gly Gly Ser Thr Asn

1 5

<210> 34

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH6 / NB27A-33 CDR3

<400> 34

Pro Glu Leu Phe Arg Leu Tyr Glu Tyr Asp Tyr

1 5 10

<210> 35

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH8 / 2-NB27A-48 CDR1

<400> 35

Ala Ser Ile Phe Ser Ile His Ala Met Gly

1 5 10

<210> 36

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH8 / 2-NB27A-48 CDR2

<400> 36

Ala Ile Thr Asn Ser Gly Ser Thr Asp

1 5

<210> 37

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH8 / 2-NB27A-48 CDR3

<400> 37

Asp Gly Arg Tyr Tyr Glu His Asp Asp Tyr

1 5 10

<210> 38

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH9 / NB26B-12 CDR1

<400> 38

Gly Phe Ile Ile Ser Asp Phe Pro Met Ser

1 5 10

<210> 39

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH9 / NB26B-12 CDR2

<400> 39

Thr Ile Thr Ser Ala Gly Gly Ser Thr Asn

1 5 10

<210> 40

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH9 / NB26B-12 CDR3

<400> 40

Asp Trp Lys Trp Tyr Glu Val Asp Asp Tyr

1 5 10

<210> 41

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH10 / 2-NB27A-10 CDR1

<400> 41

Gly Phe Ile Ile Ser Ile Tyr Ser Met Asn

1 5 10

<210> 42

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH10 / 2-NB27A-10 CDR2

<400> 42

Arg Ile Thr Arg Gly Gly Thr Thr Asn

1 5

<210> 43

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH10 / 2-NB27A-10 CDR3

<400> 43

Gly Ala Asp Gly Gly Ile

1 5

<210> 44

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH11 / 2-NB27A-120 CDR1

<400> 44

Gly Phe Thr Leu Asp Asp Asp Ala Ile Gly

1 5 10

<210> 45

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH11 / 2-NB27A-120 CDR2

<400> 45

Cys Ile Ser Ser Ser Asp Gly Ser Thr Tyr

1 5 10

<210> 46

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH11 / 2-NB27A-120 CDR3

<400> 46

Cys Gln Ile Tyr Gly Leu Gly His Asp Ala Ala Ala Pro Leu Gly Ser

1 5 10 15

<210> 47

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH12 / 2-NB27B-130 CDR1

<400> 47

Gly Phe Asn Leu Asp Tyr Tyr Ala Ile Gly

1 5 10

<210> 48

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH12 / 2-NB27B-130 CDR2

<400> 48

Cys Ile Thr Ser Ser Asp Gly Ser Thr Tyr

1 5 10

<210> 49

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH12 / 2-NB27B-130 CDR3

<400> 49

Asp Cys Met Gly Arg Glu Phe Gly Pro Ala Ser Ile

1 5 10

<210> 50

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH13 / NB27B-22 CDR1

<400> 50

Gly Phe Thr Leu Asp Tyr Tyr Gly Ile Gly

1 5 10

<210> 51

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH13 / NB27B-22 CDR2

<400> 51

Cys Ile Ser Ser Ser Asp Gly Ser Thr Tyr

1 5 10

<210> 52

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH13 / NB27B-22 CDR3

<400> 52

Ala Glu Ile Tyr Leu Ser Gly Tyr Gly Cys Tyr Val Asp Gly Ser

1 5 10 15

<210> 53

<211> 351

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH1/NB27A-15 full Length

<400> 53

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val

35 40 45

Ala Thr Val Glu Ile Gly Gly Ser Ser Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Val Asp Trp Lys Trp Tyr Glu Val Asp Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His

115 120 125

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

130 135 140

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

145 150 155 160

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

165 170 175

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

180 185 190

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

195 200 205

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

210 215 220

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

225 230 235 240

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

245 250 255

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

260 265 270

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

275 280 285

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

290 295 300

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

305 310 315 320

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

325 330 335

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

340 345 350

<210> 54

<211> 351

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH2/2-NB27B-19 full Length

<400> 54

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ser His

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Thr Arg Ser Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Thr Asp Tyr Arg Tyr Tyr Glu Ser Ile Asp Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His

115 120 125

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

130 135 140

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

145 150 155 160

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

165 170 175

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

180 185 190

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

195 200 205

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

210 215 220

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

225 230 235 240

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

245 250 255

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

260 265 270

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

275 280 285

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

290 295 300

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

305 310 315 320

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

325 330 335

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

340 345 350

<210> 55

<211> 350

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH3/2-NB27A-93 full Length

<400> 55

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Ala Ser Ile Phe Ser Ile His

20 25 30

Ala Ile Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Ser Asn Val Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Tyr Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Met Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Trp Thr Tyr Tyr Glu Val Asp Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Gln Val Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr

115 120 125

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

130 135 140

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

145 150 155 160

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

165 170 175

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

180 185 190

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

195 200 205

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

210 215 220

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

225 230 235 240

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

245 250 255

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

260 265 270

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

275 280 285

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

290 295 300

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

305 310 315 320

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

325 330 335

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

340 345 350

<210> 56

<211> 351

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH4/NB27B-6 full Length

<400> 56

Glu Val Gln Val Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Ala Ser Ile Phe Asn Ala His

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Thr Ser Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Val Asp Arg Arg Tyr Tyr Asp Leu Ala Tyr Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His

115 120 125

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

130 135 140

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

145 150 155 160

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

165 170 175

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

180 185 190

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

195 200 205

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

210 215 220

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

225 230 235 240

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

245 250 255

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

260 265 270

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

275 280 285

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

290 295 300

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

305 310 315 320

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

325 330 335

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

340 345 350

<210> 57

<211> 350

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH5/2-NB27A-95 full Length

<400> 57

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser Ile Phe Ser Ile His

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ser Ile Thr Tyr Gly Gly Asn Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Asp Asn Tyr Tyr Asp Glu Tyr Ala Pro Trp Gly Gln Gly Thr

100 105 110

Gln Val Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr

115 120 125

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

130 135 140

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

145 150 155 160

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

165 170 175

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

180 185 190

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

195 200 205

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

210 215 220

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

225 230 235 240

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

245 250 255

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

260 265 270

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

275 280 285

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

290 295 300

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

305 310 315 320

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

325 330 335

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

340 345 350

<210> 58

<211> 351

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH6/NB27A-33 full Length

<400> 58

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Gly Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Glu Tyr

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Leu Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Pro Glu Leu Phe Arg Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His

115 120 125

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

130 135 140

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

145 150 155 160

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

165 170 175

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

180 185 190

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

195 200 205

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

210 215 220

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

225 230 235 240

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

245 250 255

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

260 265 270

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

275 280 285

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

290 295 300

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

305 310 315 320

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

325 330 335

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

340 345 350

<210> 59

<211> 350

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH8/2-NB27A-48 full Length

<400> 59

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Ala Ser Ile Phe Ser Ile His

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Thr Asn Ser Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Gly Arg Tyr Tyr Glu His Asp Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Gln Val Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr

115 120 125

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

130 135 140

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

145 150 155 160

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

165 170 175

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

180 185 190

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

195 200 205

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

210 215 220

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

225 230 235 240

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

245 250 255

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

260 265 270

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

275 280 285

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

290 295 300

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

305 310 315 320

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

325 330 335

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

340 345 350

<210> 60

<211> 351

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH9/NB26B-12 full Length

<400> 60

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Ile Ser Asp Phe

20 25 30

Pro Met Ser Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Ser Ala Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Val Asp Trp Lys Trp Tyr Glu Val Asp Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His

115 120 125

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

130 135 140

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

145 150 155 160

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

165 170 175

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

180 185 190

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

195 200 205

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

210 215 220

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

225 230 235 240

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

245 250 255

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

260 265 270

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

275 280 285

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

290 295 300

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

305 310 315 320

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

325 330 335

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

340 345 350

<210> 61

<211> 346

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH10/2-NB27A-10 full Length

<400> 61

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Ile Ser Ile Tyr

20 25 30

Ser Met Asn Trp Tyr Arg Gln Ala Pro Gly Asn Glu Arg Glu Leu Val

35 40 45

Ala Arg Ile Thr Arg Gly Gly Thr Thr Asn Tyr Gly Asp Ser Val Lys

50 55 60

Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ala Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Gly Ala Asp Gly Gly Ile Trp Gly Gln Gly Thr Gln Val Thr Val

100 105 110

Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

115 120 125

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

130 135 140

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

145 150 155 160

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

165 170 175

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

180 185 190

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

195 200 205

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

210 215 220

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

225 230 235 240

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

245 250 255

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

260 265 270

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

275 280 285

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

290 295 300

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

305 310 315 320

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

325 330 335

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

340 345

<210> 62

<211> 357

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH11/2-NB27A-120 full Length

<400> 62

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Asp

20 25 30

Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Cys Ile Ser Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Thr Cys Gln Ile Tyr Gly Leu Gly His Asp Ala Ala Ala Pro Leu

100 105 110

Gly Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys

115 120 125

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

130 135 140

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

145 150 155 160

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

165 170 175

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

180 185 190

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

195 200 205

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

210 215 220

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

225 230 235 240

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

245 250 255

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

260 265 270

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

275 280 285

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

290 295 300

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

305 310 315 320

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

325 330 335

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

340 345 350

Leu Ser Pro Gly Lys

355

<210> 63

<211> 353

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH12/2-NB27B-130 full Length

<400> 63

Glu Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Leu Asp Tyr Tyr

20 25 30

Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Ile

35 40 45

Ser Cys Ile Thr Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Gly Thr Val Asn

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asp Cys Met Gly Arg Glu Phe Gly Pro Ala Ser Ile Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys

115 120 125

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

130 135 140

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

145 150 155 160

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

165 170 175

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

180 185 190

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

195 200 205

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

210 215 220

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

225 230 235 240

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

245 250 255

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

260 265 270

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

275 280 285

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

290 295 300

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

305 310 315 320

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

325 330 335

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

340 345 350

Lys

<210> 64

<211> 356

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH13/NB27B-22 full Length

<400> 64

Glu Val Arg Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr

20 25 30

Gly Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Cys Ile Ser Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Val Ala Glu Ile Tyr Leu Ser Gly Tyr Gly Cys Tyr Val Asp Gly

100 105 110

Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser

115 120 125

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

130 135 140

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

145 150 155 160

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

165 170 175

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

180 185 190

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

195 200 205

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

210 215 220

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

225 230 235 240

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

245 250 255

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

260 265 270

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

275 280 285

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

290 295 300

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

305 310 315 320

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

325 330 335

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

340 345 350

Ser Pro Gly Lys

355

<210> 65

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH1-huVH4 variable region

<400> 65

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Ser Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val

35 40 45

Ala Thr Val Glu Ile Gly Gly Ser Ser Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Val Asp Trp Lys Trp Tyr Glu Val Asp Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 66

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH2-huVH3 variable region

<400> 66

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ser His

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Thr Arg Ser Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Thr Asp Tyr Arg Tyr Tyr Glu Ser Ile Asp Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 67

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH9-huVH3 variable region

<400> 67

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Ile Ser Asp Phe

20 25 30

Pro Met Ser Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Ser Ala Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Val Asp Trp Lys Trp Tyr Glu Val Asp Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

Claims

1. An anti-IL-17A antibody or antigen-binding fragment thereof comprising a single variable domain of an immunoglobulin comprising a CDR1, CDR2, and CDR3 selected from the group consisting of:

(a-1) CDR1, the amino acid sequence of which is shown in SEQ ID NO. 17; CDR2 with amino acid sequence shown as SEQ ID NO. 18; and CDR3 with the amino acid sequence shown in SEQ ID NO. 19;

(a-2) CDR1 having the amino acid sequence shown in SEQ ID NO. 38; CDR2, its amino acid sequence is shown in SEQ ID NO 39; and CDR3 with the amino acid sequence shown as SEQ ID NO. 40;

(a-3) CDR1, the amino acid sequence of which is shown in SEQ ID NO. 20; CDR2, its amino acid sequence is shown in SEQ ID NO. 21; and CDR3 with the amino acid sequence shown as SEQ ID NO. 22;

(a-4) CDR1, the amino acid sequence of which is shown as SEQ ID NO. 23; CDR2, its amino acid sequence is shown in SEQ ID NO. 24; and CDR3 with the amino acid sequence shown as SEQ ID NO. 25;

(a-5) CDR1, the amino acid sequence of which is shown in SEQ ID NO. 35; CDR2 with the amino acid sequence shown as SEQ ID NO. 36; and CDR3 with the amino acid sequence shown as SEQ ID NO. 37;

(b-1) CDR1 having the amino acid sequence shown in SEQ ID NO. 26; CDR2, the amino acid sequence of which is shown as SEQ ID NO 27; and CDR3 with the amino acid sequence shown as SEQ ID NO. 28;

(b-2) CDR1 having the amino acid sequence shown in SEQ ID NO. 29; CDR2 with the amino acid sequence shown as SEQ ID NO. 30; and CDR3 with the amino acid sequence shown as SEQ ID NO. 31;

(b-3) CDR1 having the amino acid sequence shown in SEQ ID NO. 32; CDR2, its amino acid sequence is shown in SEQ ID NO 33; and CDR3 with the amino acid sequence shown as SEQ ID NO 34;

(b-4) CDR1 having the amino acid sequence shown in SEQ ID NO. 41; CDR2, the amino acid sequence of which is shown as SEQ ID NO. 42; and CDR3 with the amino acid sequence shown as SEQ ID NO. 43;

(c-1) CDR1 having the amino acid sequence shown in SEQ ID NO. 44; CDR2, its amino acid sequence is shown in SEQ ID NO. 45; and CDR3, its amino acid sequence is shown in SEQ ID NO 46;

(c-2) CDR1 having the amino acid sequence shown in SEQ ID NO. 47; CDR2, its amino acid sequence is shown in SEQ ID NO 48; and CDR3, its amino acid sequence is shown in SEQ ID NO 49;

(c-3) CDR1 having an amino acid sequence as shown in SEQ ID NO. 50; CDR2, its amino acid sequence is shown in SEQ ID NO. 51; and CDR3, the amino acid sequence of which is shown in SEQ ID NO. 52.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the single variable domain comprises an amino acid sequence selected from the group consisting of: SEQ ID NO. 5, SEQ ID NO. 12, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the single variable domain comprises a variant of an amino acid sequence selected from the group consisting of: SEQ ID NO. 5, SEQ ID NO. 12, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, wherein the variant has at least 80% sequence identity compared to the sequence from which it originates.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the single variable domain comprises a variant of an amino acid sequence selected from the group consisting of: SEQ ID NO. 5, SEQ ID NO. 12, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, wherein the variant has at least 85% sequence identity compared to the sequence from which it originates.

5. The antibody or antigen-binding fragment thereof of claim 1, wherein the single variable domain comprises a variant of an amino acid sequence selected from the group consisting of: SEQ ID NO. 5, SEQ ID NO. 12, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, wherein the variant has at least 90% sequence identity compared to the sequence from which it originates.

6. The antibody or antigen-binding fragment thereof of claim 1, wherein the single variable domain comprises a variant of an amino acid sequence selected from the group consisting of: SEQ ID NO. 5, SEQ ID NO. 12, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, wherein the variant has at least 91% sequence identity compared to the sequence from which it originates.

7. The antibody or antigen-binding fragment thereof of claim 1, wherein the single variable domain comprises a variant of an amino acid sequence selected from the group consisting of: SEQ ID NO. 5, SEQ ID NO. 12, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, wherein the variant has at least 92% sequence identity compared to the sequence from which it originates.

8. The antibody or antigen-binding fragment thereof of claim 1, wherein the single variable domain comprises a variant of an amino acid sequence selected from the group consisting of: SEQ ID NO. 5, SEQ ID NO. 12, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, wherein the variant has at least 93% sequence identity compared to the sequence from which it originates.

9. The antibody or antigen-binding fragment thereof of claim 1, wherein the single variable domain comprises a variant of an amino acid sequence selected from the group consisting of: SEQ ID NO. 5, SEQ ID NO. 12, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, wherein the variant has at least 94% sequence identity compared to the sequence from which it originates.

10. The antibody or antigen-binding fragment thereof of claim 1, wherein the single variable domain comprises a variant of an amino acid sequence selected from the group consisting of: SEQ ID NO. 5, SEQ ID NO. 12, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, wherein the variant has at least 95% sequence identity compared to the sequence from which it originates.

11. The antibody or antigen-binding fragment thereof of claim 1, wherein the single variable domain comprises a variant of an amino acid sequence selected from the group consisting of: SEQ ID NO. 5, SEQ ID NO. 12, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, wherein the variant has at least 96% sequence identity compared to the sequence from which it originates.

12. The antibody or antigen-binding fragment thereof of claim 1, wherein the single variable domain comprises a variant of an amino acid sequence selected from the group consisting of: SEQ ID NO. 5, SEQ ID NO. 12, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, wherein the variant has at least 97% sequence identity compared to the sequence from which it originates.

13. The antibody or antigen-binding fragment thereof of claim 1, wherein the single variable domain comprises a variant of an amino acid sequence selected from the group consisting of: SEQ ID NO. 5, SEQ ID NO. 12, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, wherein the variant has at least 98% sequence identity compared to the sequence from which it originates.

14. The antibody or antigen-binding fragment thereof of claim 1, wherein the single variable domain comprises a variant of an amino acid sequence selected from the group consisting of: SEQ ID NO. 5, SEQ ID NO. 12, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, wherein the variant has at least 99% sequence identity compared to the sequence from which it originates.

15. The antibody or antigen-binding fragment thereof of claim 1, wherein the single variable domain comprises an amino acid sequence selected from the group consisting of: SEQ ID NO. 65, SEQ ID NO. 67 and SEQ ID NO. 66.

16. The antibody or antigen-binding fragment thereof of any one of claims 1-15, which is a single domain antibody, heavy chain antibody, humanized antibody, or chimeric antibody.

17. The antibody or antigen-binding fragment thereof of any one of claims 1-15, further comprising an Fc fragment of an immunoglobulin.

18. The antibody or antigen-binding fragment thereof of claim 17, wherein the Fc fragment is an Fc fragment of human IgG 1.

19. The antibody or antigen-binding fragment thereof of claim 17, wherein the Fc fragment comprises the amino acid sequence of SEQ ID No. 2.

20. The antibody or antigen-binding fragment thereof of any one of claims 1-15, which is capable of

1) Specifically binds IL-17A;

2) Neutralizing IL-17A;

3) Specifically binds to IL-17A homodimers or IL-17A/F heterodimers; and/or

4) Blocking the interaction of IL-17A or IL-17A/F with IL-17 RA.

21. The antibody or antigen-binding fragment thereof of any one of claims 1-15, conjugated to at least one detectable label.

22. The antibody or antigen-binding fragment thereof of claim 21, wherein the detectable label is selected from the group consisting of a radioisotope, biotin, colloidal gold, and a fluorescent group.

23. A multispecific antibody comprising a first antigen-binding portion that binds IL-17A and a second antigen-binding portion that binds a second antigen, wherein the first antigen-binding portion comprises the antibody or antigen-binding fragment thereof of any one of claims 1-22.

24. A polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1-20.

25. An expression vector comprising the polynucleotide of claim 24.

26. A host cell comprising the polynucleotide of claim 24 or the expression vector of claim 25.

27. A method of making the anti-IL-17A antibody or antigen-binding fragment thereof of any one of claims 1-20, comprising culturing the host cell of claim 26 under suitable conditions to express the antibody or antigen-binding fragment thereof, and isolating the antibody or antigen-binding fragment thereof from the host cell or culture thereof.

28. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-22, and a pharmaceutically acceptable carrier.

29. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-22 or the pharmaceutical composition of claim 28 in the manufacture of a medicament for treating an IL-17A mediated disease, wherein the IL-17A mediated disease is selected from the group consisting of Rheumatoid Arthritis (RA), psoriasis, crohn's disease, multiple Sclerosis (MS), asthma, and lupus erythematosus.

30. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-22 in the preparation of a kit for detecting the presence or determining the expression level of IL-17A in a sample.