KR102048475B1 - Antibodies Specifically Binding Against IL-17A and Uses Thereof - Google Patents

Abstract

The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to IL-17A, a polynucleotide encoding the same, a vector comprising the polynucleotide, a cell transformed with the vector, the preparation of the antibody or antigen-binding fragment thereof. A method, an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof, a double antibody comprising the antibody or antigen-binding fragment thereof, and a composition for preventing or treating cancer or autoimmune disease thereof.

Description

Antibodies Specific Binding Against IL-17A and Uses Thereof}

The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to IL-17A and does not exhibit binding to IL-17F, a polynucleotide encoding the same, a vector comprising the polynucleotide, a cell transformed with the vector, A method for producing an antibody or antigen-binding fragment thereof, an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof, a double antibody comprising the antibody or antigen-binding fragment thereof and a composition for preventing or treating cancer or autoimmune disease thereof It is about.

IL-17 was discovered in 1993 as a human cytotoxic T lymphocyte-associated antigen 8 (CTLA8), and in 1995, IL-17RA was discovered and named Interleukin 17. IL-17A forms homodimers or heterodimers with IL-17F, and IL-17F homodimers are also present. (Nat Rev Immunol. 2009 Aug; 9 (8): 556-67).

Among them, IL-17A is secreted from immune cells, such as Th17 and CD8 cells, to induce autoimmune pathology, neutrophil recruitment, G-CSF (granulocyte colony-stimulating factor), IL-6, IL It is thought to be an alternative to TNF-α inhibitors by promoting the secretion of pro-inflammatory cytokines such as -8.

IL-17A has a very high and even binding capacity of Kd 2.8 ± 0.9 nM to IL-17RA and Kd 1.2 ± 0.1 nM to IL-17RC, whereas IL-17F is highly sensitive to receptors IL-17RA and IL-17RC. Since the binding capacity of Kd 292 ± 19 nM and Kd 4.4 ± 0.2 nM, respectively, the binding capacity of IL-17A to the receptor is about 100 times higher than that of the IL-17F receptor (Nat Immunol. 2009 Dec 10 (12): 1245-51). IL-17A has a higher expression level than IL-17F and its activity of inducing inflammation with IL-17F alone is lower than that of IL-17A. However, IL-17A, like IL-17A, is known to cause a synergistic response with TNF-α. .

However, since IL-17F is an inflammatory disease factor like IL-17A but is also a major component of host defense against extracellular pathogens and microbial infections, treatment for IL-17F inhibition must be careful and controversial ( Immunity. 2011 Feb 25; 34 (2): 149-62).

Previous reports suggest that IL-17A specific antibodies developed for the treatment of autoimmune diseases, including rheumatoid arthritis (RA), psoriasis and psoriatic arthritis (PsA), reduce IL-17A levels in the body of patients, It is known to indicate significant relief of symptoms. However, anti-IL-17A treatments, cecukinumab, ixekizumab, and brodalumab (anti-IL17RA), show differences in pharmacological activity among patients (Ann Rheum Dis. 2013 Apr; 72 Suppl 2: ii116-23), which necessitates discussion of the appropriate dose of the drug and the possible side effects. There is a difference in the efficacy of drugs between patients treated with anti-IL-17A, which may be related to the expression patterns of IL-17A and IL-17F and the heterodimer IL-17A / F. Presented. Indeed, this study reported that IL-17A in RA and PsA patients showed similar patterns, but differences in expression of IL-17F and receptors in some patients (Arthritis Res Ther. 2014 Aug 22; 16 (4). ): 426).

The inventors of the present application have confirmed that although Secukinumab and the like previously developed are IL-17A specific antibodies, they have binding ability to IL-17F. Considering the possibility of causing Under these technical backgrounds, the inventors of the present application have made diligent efforts to develop an antibody that specifically binds to IL-17A, and as a result, develop an antibody that binds only to IL-17A without high binding to IL-17F, It was confirmed that such antibodies can reduce the occurrence of side effects while showing the desired therapeutic effect, and completed the present invention.

The above information described in this Background section is only for improving the understanding of the background of the present invention, and therefore does not include information that forms a prior art known to those of ordinary skill in the art. You may not.

An object of the present invention is to provide a novel antibody or antigen-binding fragment thereof that specifically binds IL-17A.

Another object of the present invention is to provide a polynucleotide encoding the antibody or antigen-binding fragment thereof.

Another object of the present invention is to provide a vector comprising the polynucleotide, a cell transformed with the vector, and a method of manufacturing the same.

Another object of the present invention is to provide an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof.

Another object of the present invention is to provide a double antibody comprising the antibody or antigen-binding fragment thereof.

Still another object of the present invention is to provide a composition for preventing or treating cancer or autoimmune disease, including the antibody or antigen-binding fragment thereof.

In order to achieve the above object, the present invention provides a heavy chain CDR1 of SEQ ID NO: 1 or SEQ ID NO: 8; A heavy chain CDR2 selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 9, and SEQ ID NO: 11; And a heavy chain CDR3 selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 10, and light chain CDR1 of SEQ ID NO: 12; Light chain CDR2 of SEQ ID NO: 13; And an antibody or antigen-binding fragment thereof that specifically binds to IL-17A comprising the light chain CDR3 of SEQ ID NO: 14.

The present invention also provides a polynucleotide encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof.

The present invention also provides a vector comprising the polynucleotide.

The present invention also provides a cell transformed with the vector.

The present invention also provides a method of preparing the antibody or antigen-binding fragment thereof, comprising the steps of: (a) culturing the cells; And (b) recovering the antibody or antigen-binding fragment thereof from the cultured cells.

The present invention also provides an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof.

The present invention also provides a double antibody comprising the antibody or antigen-binding fragment thereof.

The present invention further provides a composition for preventing or treating cancer or autoimmune disease comprising the antibody or antigen-binding fragment thereof, or administering the antibody or antigen-binding fragment thereof to a subject. Provide prevention or treatment.

The antibody or antigen-binding fragment thereof according to the present invention exhibits high specificity for IL-17A, and thus can act only on IL-17A of a patient to derive a constant and stable therapeutic effect between patients. In addition, since only the IL-17A axis is inhibited, various side effects such as blood cancers such as lymphoma, central nervous system diseases, and skin diseases including rashes may occur, which may be caused by affecting the surrounding normal immune system. I expect to be able to lower things.

1 is a schematic diagram of hIL-17A and mIL-17A expression vectors in which His-tag is fused to the carboxy-terminus.
Figure 2a shows the expression results of hIL-17A-His and mIL-17A-His in the temporary expression system, Figure 2b shows the results of purification of hIL-17A-His and mIL-17A-His.
Figure 3 shows the binding force of positive phage to human IL-17A according to the number of panning.
Figure 4 shows the positive binding signal for IL-17His of single scFv-phage clones through ELISA results.
Figure 5 shows a schematic diagram and the results of analyzing the IL-17A Ligand-Receptor binding inhibitory ability of the candidate antibody through a competitive ELISA.
Figure 6 is a result of comparing the binding inhibition of the candidate antibody through the IL-17A Ligand-Receptor binding inhibition assay.
7 is a schematic diagram and result of evaluating whether IL-6 secretion dependent on IL-17A was inhibited by antibody using HeLa cell line.
Figure 8 shows a schematic diagram of preparing the complementarity determining region variants of the heavy chain variable region of 7D1 and 9E12.
9 is a result of analyzing the IL-17A Ligand-Receptor binding inhibitory ability of the antibody derived through the complementarity determining site changes.
10 shows the results of SDS-PAGE analysis for confirming the purity of IL-17A antibodies 7H3C11FW and 9H2D9.
11A, 11B and 11C each show ELISA results showing that the antibodies derived through optimization show specific binding to IL-17A and no non-specific binding.
Figure 12 shows the results showing that the antibody according to the present invention can inhibit the concentration gradient dependent IL-6 secreted IL-17A dependent.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention relates to an antibody or antigen-binding fragment thereof, which specifically binds to IL-17A and does not bind to IL-17F. The antibody or antigen-binding fragment thereof that specifically binds to IL-17A according to the present invention shows that the antibody does not bind to human IL-17F at all through non-specific ELISA and specific ELISA. It was confirmed that it showed superior specificity that specifically binds only to IL-17A.

Based on this, the present invention provides a heavy chain CDR1 of SEQ ID NO: 1 or SEQ ID NO: 8; A heavy chain CDR2 selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 9, and SEQ ID NO: 11; And a heavy chain CDR3 selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 10, and light chain CDR1 of SEQ ID NO: 12; Light chain CDR2 of SEQ ID NO: 13; And an antibody or antigen-binding fragment thereof that specifically binds IL-17A comprising the light chain CDR3 of SEQ ID NO: 14.

IL-17A herein is, for example, human IL-17A. Human IL-17A according to the present invention may comprise, for example, the sequence of SEQ ID NO: 58. The inventors of the present application used phage display technology to select antibodies that bind only to IL-17A with high affinity from the human antibody library.

Antibodies or antigen-binding fragments thereof according to the invention specifically bind only to IL-17A, do not bind to IL-17F, for example for IL-17A, for example, 1.0 × 10 ⁻⁸ M or less, 1.0 × 10 ⁻⁹ A binding affinity (KD) of M or less, 1.0X10 ^-10 M or less, 1.0X10 ^-11 M or less, 1.0X10 ^-12 M or less. Antibodies or antigen-binding fragments thereof according to the present invention may exhibit a binding affinity (KD) outside the ranges defined above for IL-17F, which may be considered to exhibit no binding capacity to IL-17F.

As used herein, the term "antibody" refers to an anti-IL-17A antibody that specifically binds to IL-17A. The scope of the present invention includes not only complete antibody forms that specifically bind to IL-17A, but also antigen binding fragments of such antibody molecules.

A complete antibody is a structure having two full length light chains and two full length heavy chains, each of which is linked by heavy and disulfide bonds. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types and subclasses gamma 1 (γ1), gamma 2 (γ2), and gamma 3 (γ3). ), Gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant regions of the light chains have kappa (κ) and lambda (λ) types. The basic four-chain antibody unit is a heterodimeric glycoprotein consisting of two identical light chains (L) and two identical heavy chains (H). The light chain has a variable region (VL) at the N-terminus, followed by a constant region at its other end. The heavy chain has a variable region (VH) at the N-terminus, followed by three constant regions (CH) for the α and γ chains and four CH regions for the μ and ε isotypes, respectively. Variables indicate that certain portions of the variable region differ greatly in sequence between antibodies. The V region mediates antigen binding and defines the specificity of a particular antibody for that particular antigen. Variability is concentrated in three segments called hypervariable regions (HVRs), or CDRs, in both the light and heavy chain variable regions. The more highly conserved portions of the variable regions are called framework regions (FRs). The heavy and light chain variable regions have the structures FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from the N-terminus to the C-terminus.

As used herein, the term “heavy chain” refers to a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen and a full length heavy chain comprising three constant region domains CH1, CH2 and CH3 And fragments thereof.

In addition, the term "light chain" as used herein refers to a full-length light chain and fragment thereof comprising a variable region domain VL and a constant region domain CL comprising an amino acid sequence having sufficient variable region sequence to confer specificity to the antigen. All means.

An antigen binding fragment or antibody fragment of an antibody means a fragment having an antigen binding function and includes Fab, F (ab ') ₂ and Fv.

"Fv" fragments are antibody fragments containing complete antibody recognition and binding sites. This region consists of a dimer in which one heavy chain variable domain and one light chain variable domain are tightly and covalently associated, for example, with scFv.

"Fab" fragments contain the variable and constant domains of the light chain and the variable and first constant domains (CH1) of the heavy chain. F (ab ') ₂ antibody fragments generally comprise a pair of Fab fragments covalently linked near their carboxy termini by hinge cysteines between them.

"Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, which domains are present in a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH domain and the VL domain that allows the scFv to form the desired structure for antigen binding.

In one embodiment, the antibody according to the invention is in Fv form (eg scFv) or is in the form of a complete antibody. In addition, the heavy chain constant region may be selected from any one isotype of gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε). For example, the constant region is gamma 1 (IgG1), gamma 3 (IgG3) or gamma 4 (IgG4). The light chain constant region may be of kappa or lambda type.

Antibodies of the invention are monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fvs (scFV), single chain antibodies, Fab fragments, F (ab ') fragments, disulfide-binding Fvs (sdFV) And anti-idiotype (anti-Id) antibodies, or epitope-binding fragments of the antibodies, and the like.

Said monoclonal antibody refers to the same except for possible naturally occurring mutations in which antibodies obtained from substantially homogeneous antibody populations, ie, individual antibodies in the population, may be present in trace amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site.

Non-human (eg murine) antibodies of the “humanized” form are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. In most cases, humanized antibodies are non-human species (donor antibodies) that retain the desired specificity, affinity, and capacity for residues from the hypervariable region of the recipient, for example mice, rats, rabbits, or non-humans. Human immunoglobulins (receptor antibodies) replaced with residues from the hypervariable regions of primates.

The term "human antibody" refers to a molecule derived from human immunoglobulin, in which all of the amino acid sequences constituting the antibody including complementarity determining regions and structural regions are composed of human immunoglobulins. Human antibodies can be prepared using various techniques known in the art, including phage display libraries. Human antibodies have been modified to produce antibodies in response to antigen test inoculation, but can be made by administering the antigen to a transgenic animal, eg, an immunized xenomouse, whose endogenous locus does not function. Whereas conventional ixekizumab is a humanized antibody, the antibody according to the present invention is a human antibody.

While the heavy and / or light chain portions are the same or homologous to the corresponding sequences in an antibody derived from a particular species or belonging to a particular antibody class or subclass, the remaining chain (s) are derived from another species or another antibody class or Included are "chimeric" antibodies (immunoglobulins) that are identical or homologous to the corresponding sequences in antibodies belonging to the subclass, as well as fragments of such antibodies that exhibit the desired biological activity.

"Antibody variable domain" as used herein refers to the light and heavy chain portions of an antibody molecule comprising the amino acid sequences of complementarity determining regions (CDRs; ie CDR1, CDR2, and CDR3), and framework regions (FR) do. VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

"Complementarity determining regions" (CDRs; ie CDR1, CDR2, and CDR3) refer to amino acid residues of an antibody variable domain that are necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3.

In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, a heavy chain CDR3 of SEQ ID NO: 3; Heavy chain CDR1 of SEQ ID NO: 1, heavy chain CDR2 of SEQ ID NO: 4, heavy chain CDR3 of SEQ ID NO: 5; Heavy chain CDR1 of SEQ ID NO: 1, heavy chain CDR2 of SEQ ID NO: 4, heavy chain CDR3 of SEQ ID NO: 6; Heavy chain CDR1 of SEQ ID NO: 1, heavy chain CDR2 of SEQ ID NO: 4, heavy chain CDR3 of SEQ ID NO: 7; Heavy chain CDR1 of SEQ ID NO: 8, heavy chain CDR2 of SEQ ID NO: 9, heavy chain CDR3 of SEQ ID NO: 10; Or heavy chain CDR1 of SEQ ID NO: 8, heavy chain CDR2 of SEQ ID NO: 11, heavy chain CDR3 of SEQ ID NO: 10.

"Framework regions" (FRs) are variable domain residues other than CDR residues. Each variable domain typically has four FRs identified as FR1, FR2, FR3 and FR4.

In one embodiment, the antibody or antigen-binding fragment thereof according to the invention may comprise a heavy chain variable region framework (FR) selected from the group consisting of SEQ ID NO: 15 to SEQ ID NO: 26. Specifically, FR1 of SEQ ID NO: 15, FR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and FR4 of SEQ ID NO: 18; FR1 of SEQ ID NO: 19, FR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 20, and FR4 of SEQ ID NO: 21; FR1 of SEQ ID NO: 22, FR2 of SEQ ID NO: 23, FR3 of SEQ ID NO: 24, and FR4 of SEQ ID NO: 25; And a heavy chain variable region framework selected from the group consisting of FR1 of SEQ ID NO: 22, FR2 of SEQ ID NO: 26, FR3 of SEQ ID NO: 24, and FR4 of SEQ ID NO: 25.

In another embodiment, the antibody or antigen-binding fragment thereof according to the present invention may comprise a light chain variable region framework (FR) selected from the group consisting of SEQ ID NO: 27 to SEQ ID NO: 33. Specifically, FR1 of SEQ ID NO: 27, FR2 of SEQ ID NO: 28, FR3 of SEQ ID NO: 29, and FR4 of SEQ ID NO: 30; Or a light chain variable region framework of FR1 of SEQ ID NO: 31, FR2 of SEQ ID NO: 32, FR3 of SEQ ID NO: 33, and FR4 of SEQ ID NO: 30.

Including the CDR1 to CDR3 and the framework, the antibody or antigen-binding fragment thereof according to the present invention may include, for example, a heavy chain variable region selected from the group consisting of SEQ ID NO: 34 to SEQ ID NO: 39. In addition, the antibody or antigen-binding fragment thereof according to the present invention may include, for example, the light chain variable region of SEQ ID NO: 40 or SEQ ID NO: 41.

In one embodiment, the antibody or antigen-binding fragment thereof of the invention may comprise the following heavy chain variable region and light chain variable region:

(Iii) the heavy chain variable region of SEQ ID NO: 34 and the light chain variable region of SEQ ID NO: 40;

(Ii) a heavy chain variable region of SEQ ID NO: 35 and a light chain variable region of SEQ ID NO: 41;

(Iii) the heavy chain variable region of SEQ ID NO: 36 and the light chain variable region of SEQ ID NO: 41;

(iv) a heavy chain variable region of SEQ ID NO: 37 and a light chain variable region of SEQ ID NO: 41;

(v) a heavy chain variable region of SEQ ID NO: 38 and a light chain variable region of SEQ ID NO: 40; or

(vi) the heavy chain variable region of SEQ ID NO: 39 and the light chain variable region of SEQ ID NO: 41.

"Phase display" is a technique for displaying variant polypeptides as phage proteins, such as fusion proteins with at least a portion of the envelope protein on the surface of fibrous phage particles. The utility of phage display lies in the fact that a large library of randomized protein variants can be targeted to quickly and efficiently classify sequences that bind with high affinity with a target antigen. Displaying peptide and protein libraries on phage has been used to screen millions of polypeptides to identify polypeptides with specific binding properties.

Phage display technology provided a powerful tool for generating and selecting new proteins that bind specific ligands (eg antigens). Phage display technology can be used to generate large libraries of protein variants and to quickly sort sequences that bind with high affinity to target antigens. Nucleic acids encoding variant polypeptides are fused with nucleic acid sequences encoding viral envelope proteins, eg, gene III protein or gene VIII protein. Monovalent phage display systems have been developed in which a nucleic acid sequence encoding a protein or polypeptide is fused with a nucleic acid sequence encoding a portion of a gene III protein. In monovalent phage display systems, gene fusions are expressed at low levels and wild type Gene III proteins are also expressed to maintain particle infectivity.

Demonstrating the expression of peptides on fibrous phage surfaces and the expression of functional antibody fragments in the periplasm of E. coli is important in developing antibody phage display libraries. Libraries of antibody or antigen-binding polypeptides have been prepared in a number of ways, for example by altering a single gene by inserting random DNA sequences or by cloning related gene families. Libraries can be screened for expression of antibodies or antigen binding proteins with the desired characteristics.

Phage display technology has several advantages over conventional hybridoma and recombinant methods for preparing antibodies with the desired characteristics. This technique allows the production of large antibody libraries with various sequences in a short time without the use of animals. The preparation of hybridomas or the production of humanized antibodies may require months of preparation. In addition, since no immunity is required at all, phage antibody libraries can produce antibodies against antigens that are toxic or low antigenic. Phage antibody libraries can also be used to generate and identify novel therapeutic antibodies.

Techniques for generating human antibodies from immunized, non-immunized human, germline sequences, or naïve B cell Ig repertory using immunized phage display libraries can be used. Various lymphoid tissues can be used to prepare naïve or non-immune antigen binding libraries.

The ability to identify and isolate high affinity antibodies from phage display libraries is important for the isolation of novel therapeutic antibodies. Separation of high affinity antibodies from the library may depend on the size of the library, the efficiency of production in bacterial cells, and the diversity of the library. The size of the library is reduced by inefficient folding of the antibody or antigen binding protein and inefficient production due to the presence of stop codons. Expression in bacterial cells can be inhibited if the antibody or antigen binding domain is not properly folded. Expression can be improved by alternating mutations at the surface of the variable / constant interface or at selected CDR residues. The sequence of the backbone region is one element to provide proper folding when generating antibody phage libraries in bacterial cells.

It is important to generate various libraries of antibodies or antigen binding proteins in high affinity antibody isolation. CDR3 regions have been found to often participate in antigen binding. The CDR3 regions on the heavy chains vary considerably in size, sequence, and structural conformation, and thus can be used to prepare a variety of libraries.

In addition, diversity can be generated by randomizing the CDR regions of the variable heavy and light chains using all 20 amino acids at each position. The use of all twenty amino acids can result in highly variable variant antibody sequences and increase the chance of identifying new antibodies.

As long as the antibody or antibody fragment of the present invention is maintained, antibodies or antibody fragments in which mutations in the variable region have occurred are also included in the scope of the present invention. An example is an antibody in which a conservative substitution of amino acids in a variable region occurs. Conservative substitutions mean substitutions with other amino acid residues that have properties similar to those of the original amino acid sequence. For example, lysine, arginine, and histidine have similar properties because they have a base side chain. Aspartic acid and glutamic acid It has similar characteristics in that it has a mountain side chain. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar characteristics in that they have a non-charged polar side chain, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine are nonpolar. Tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have side chains. Therefore, it will be apparent to those skilled in the art that amino acid substitutions within a group having similar characteristics as described above will not show any change in characteristics, so as long as the characteristics of the antibody according to the present invention are maintained, Antibodies that have undergone mutations due to conservative substitutions in are also included in the scope of the present invention.

Considering the above-described variations with biologically equivalent activity, an antibody or antibody fragment of the present invention, or a polynucleotide encoding the same, is interpreted to also include a sequence that exhibits substantial identity with the sequence set forth in SEQ ID NO. The above substantial identity is at least 61% when the sequence of the present invention is aligned as closely as possible with any other sequence, and the aligned sequence is analyzed using algorithms commonly used in the art. Homology, more preferably 70% homology, even more preferably 80% homology, most preferably 90% homology, preferably at least 95%, at least 96%, at least 97%, 98% Or more than 99% of the same sequence. Alignment methods for sequence comparison are known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NBCI and the like and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn and tblastx on the Internet. BLSAT is accessible at www.ncbi.nlm.nih.gov/BLAST/. Sequence homology comparisons using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

In another aspect, the invention relates to a polynucleotide encoding said antibody or antigen binding fragment thereof.

Polynucleotides encoding an antibody or antigen-binding fragment thereof of the invention can be recombinantly produced for the antibody or antigen-binding fragment thereof. The polynucleotide nucleic acid is isolated and inserted into a replicable vector for further cloning (amplification of DNA) or for further expression. Based on this, the present invention relates to a vector comprising the polynucleotide in another aspect.

"Polynucleotide" is meant to encompass a plurality of nucleic acids, such as DNA (gDNA and cDNA) and RNA molecules, and the nucleotides, which are the basic structural units in nucleic acids, are modified not only in the natural nucleotides, but also in sugar or base sites. Also included are analogues. The sequence of polynucleotides encoding heavy and light chain variable regions of the invention can be modified. Such modifications include addition, deletion, or non-conservative or conservative substitutions of nucleotides.

In one embodiment, the polynucleotide encoding the antibody or antigen binding site thereof according to the invention may be selected from the group consisting of, for example, SEQ ID NO: 42 to SEQ ID NO: 49. Specifically, the polynucleotide encoding the heavy chain variable region may be selected from the group consisting of SEQ ID NO: 42 to SEQ ID NO: 47. In addition, the polynucleotide encoding the light chain variable region may be SEQ ID NO: 48 or SEQ ID NO: 49.

Nucleic acids of the invention are also construed to include nucleotide sequences that exhibit substantial identity to the nucleotide sequence. Substantial identity is at least 80% homology when aligning the nucleotide sequence of the present invention with any other sequence as closely as possible and analyzing the aligned sequence using algorithms commonly used in the art. Preferably a nucleotide sequence that exhibits at least 90% homology, most preferably at least 95% homology, preferably at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology do.

The DNA encoding the antibody can be readily isolated or separated using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the DNA encoding the heavy and light chain variable regions of the antibody). Synthesize Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: signal sequence, origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences.

As used herein, the term "vector" refers to a plasmid vector as a means for expressing a gene of interest in a host cell; Cosmid vector; Viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors, and the like. The nucleic acid encoding the antibody in the vector is operably linked with a promoter.

"Operatively linked" means a functional binding between a nucleic acid expression control sequence (eg, an array of promoters, signal sequences, or transcriptional regulator binding sites) and another nucleic acid sequence, whereby the regulatory sequence is the other nucleic acid. To control transcription and / or translation of the sequence.

In the case of a prokaryotic cell as a host, powerful promoters capable of promoting transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), ribosome binding sites for initiation of translation, and transcription / detox termination sequences. Also, for example, when the eukaryotic cell is a host, a promoter derived from the genome of the mammalian cell (e.g., a metallothionine promoter, a β-actin promoter, a human heroglobin promoter and a human muscle creatine promoter) or a mammal Promoters derived from animal viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse breast tumor virus (MMTV) promoter, LTR promoter of HIV) , Promoter of the Moroni virus Epsteinbar virus (EBV) and Loose Sacoma virus (RSV) promoter) can be used, and generally has a polyadenylation sequence as a transcription termination sequence.

In some cases, the vector may be fused with other sequences to facilitate purification of the antibody expressed therefrom. Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA).

Such vectors include antibiotic resistance genes commonly used in the art as selectable markers and include, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline. There is a resistance gene.

In another aspect, the present invention relates to a cell transformed with the above-mentioned vector. The cells used to produce the antibodies of the invention can be prokaryote, yeast or higher eukaryote cells, but are not limited thereto.

Bacillus strains such as Escherichia coli, Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g. Pseudomonas putida), Proteus Prokaryotic host cells such as Proteus mirabilis and Staphylococcus (eg, Staphylocus carnosus) can be used.

However, the greatest interest in animal cells, examples of useful host cell lines are COS-7, BHK, CHO, CHO-S, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0 , NS-0, U20S, or HT1080, but is not limited thereto.

In another aspect, the present invention, (a) culturing the cells; And (b) recovering the antibody or antigen-binding fragment thereof from the cultured cells.

The cells can be cultured in various media. It can be used as a culture medium without limitation among commercial media. All other necessary supplements known to those skilled in the art may be included at appropriate concentrations. Culture conditions, such as temperature, pH, and the like, are already in use with host cells selected for expression, which will be apparent to those skilled in the art.

The recovery of the antibody or antigen-binding fragment thereof can be removed by, for example, centrifugation or ultrafiltration, and the resultant can be purified using, for example, affinity chromatography or the like. Further other purification techniques such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography and the like can be used.

The present invention also relates to an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof and a drug bound thereto.

Such drugs include chemicals, radionuclides, immunotherapeutic agents, cytokines, chemokines, toxins, biological agents and enzyme inhibitors. For example, an antibody or fragment thereof of the present invention may be directly or indirectly bound to an anticancer agent, and examples of the anticancer agent include acibaicin, aclarubicin, acodazole, acronycin, adozelesin, alanosine, Aldesleukin, Allopurinol Sodium, Altretamine, Aminoglutetimide, Amonapiide, Ampligen, Amsacrine, Androgens, Anguidine, Apidicholine glycinate, Asari, Asparaginase, 5-Azacytidine, Azathioprine, Bacillus Calmette-Guerin (BCG), Bakers Antipol, Beta-2-Dioxythioguanosine, Bisantrene HCl, Bleomycin Sulfate, Bullpanol, Butionine Sulfoximine, BWA773U82, BW 502U83 / HCl, BW 7U85 mesylate, cerasemide, carbetimer, carboplatin, carmustine, chlorambucil, chloroquinoxaline-sulfonamide, chlorozotocin, chromomycin A3, Cisplatin, Cladribine, Cortico Steroids, corinbacterium parboom, CPT-11, cristolol, cyclocytidine, cyclophosphamide, cytarabine, cytembena, davis maleate, decarbazine, dactinomycin, daunorubicin HCl, Diazauridine, dexrazoic acid, dianhydrogalactitol, diazzione, dibromodulitol, dididemin B, ethyldithiocarbamate, diclicoaldehyde, dihydro-5-azacytin, doxorubicin, Echinomycin, Dedatrexate, Edelfosine, Eprolnitine, Eliots Solution, Elsamitrucin, Epirubicin, Esorubicin, Estramustine Phosphate, Estrogen, Etanidazole, Ethiophos, Etoposide, Parfait Drazole, pazarabine, fenretinide, filgrastim, finasteride, flavone acetic acid, phloxuridine, fludarabine phosphate, 5'-fluorouracil, Fluosol ™, flutamide, gallium nitrile , Gemcitabine, goserelin acetate, hepsulfam, hexamethylene bisacetamide, homoharingtonin, hydrazine sulfate, 4-hydroxyandrostenedione, hydrourea, idarubicin HCl, ifosfamide, 4- Ipomeanol, Iproplatin, Isotretinoin, Leucovorin Calcium, Leuprolide Acetate, Levamisol, Liposome Daunorubicin, Liposome Capture Doxorubicin, Romestin, Rodinamine, Maytansine, Mechloretamine Hydrochloride, Melphalan, Methanol, merbaron, 6-mercaptopurine, mesna, methanol extract of Bacillus calete-guerine, methotrexate, N-methylformamide, mifepristone, mitoguazone, mitomycin-C, mitotan, mitoxane Tron Hydrochloride, Monosite / Macrophage Colony-Stimulating Factor, Nabilone, Napoxidine, Neocarcinostatin, Octreotide Acetate, Or Platin, oxaliplatin, paclitaxel, pala, pentostatin, piperazinedione, fibrobromine, pyrarubicin, pyritrexime, pyroxanthrone hydrochloride, PIXY-321, plymycin, porpimer sodium, prednimu Styrene, Procarbazine, Progestins, Pyrazopurin, Lazoic Acid, Sargramothim, Semustine, Spirogermanium, Spyromustine, Streptonigreen, Streptozosin, Sulfenerner, Suramin Sodium, Tamoxifen, Taxorere , Tegapur, teniposide, terephthalamidine, teroxylone, thioguanine, thiotepa, thymidine injection, thiazopurin, topotecan, toremifene, tretinoin, trifluoroperazine hydrochloride, trifluidine, trimeth Trexate, TNF (tumor necrosis factor), uracil mustard, vinblastine sulphate, vincristine sulphate, vindesine, vinorelbine, vinzolidine, Yoshi 864, zorubicin, cytosine Lavinoside, etoposide, melparan, taxotel, taxol and the like.

The present invention also relates to a double antibody comprising the antibody or antigen-binding fragment thereof and a drug bound thereto.

The double antibody is an antibody capable of binding to two different kinds of antigens (target proteins), and may be in a form prepared by genetic engineering or any method. The dual antibody according to the present invention may be in the form of a plurality of antibodies linked by a linker.

The linker can connect two different fusion partners (eg, biological polymers) using hydrogen bonds, electrostatic interactions, van der Waals forces, disulfide bonds, salt bridges, hydrophobic interactions, covalent bonds, and the like. Conjugates, specifically, having at least one cysteine capable of participating in at least one disulfide bond under physiological conditions or other standard peptide conditions (eg, peptide purification conditions, peptide storage conditions), In addition to simply connecting the respective fusion partners, it may serve to provide a certain sized gap between the fusion partners, or may serve as a hinge that provides flexibility or rigidity to the fusion. The linker may be a non-peptide linker or a peptide linker, and may include all directly linked by peptide bonds, disulfide bonds, and the like.

The peptide linker may comprise an amino acid sequence in the form of a plurality of amino acid sequences or motifs repeated.

The non-peptide linkers include polyethylene glycol (PEG) homopolymers, polypropylene glycol homopolymers, ethylene glycol-propylene glycol copolymers, polyoxy ethylated polyols, polyvinyl alcohols, polysaccharides, dextran, polyvinyl ethyl Biodegradable polymers such as ethers, lipid polymers, chitins, hyaluronic acid or combinations thereof. Specifically, it may be a polyethylene glycol homopolymer, and derivatives thereof known in the art and derivatives which can be easily prepared at the technical level in the art may be included in the scope of the present invention.

Sites directly or indirectly linked through the linker are not particularly limited, but may be Fc moieties, Fab ′, F (ab ′) 2, Fab, Fv, and the like.

In another aspect, the present invention relates to a composition for preventing or treating autoimmune diseases, comprising the antibody or antigen-binding fragment thereof, or the antibody-drug conjugate as an active ingredient.

The present invention comprises, for example, (a) a pharmaceutically effective amount of an antibody or antigen-binding fragment thereof according to the invention, or said antibody-drug conjugate; And (b) may be a pharmaceutical composition for the prevention or treatment of autoimmune diseases comprising a pharmaceutically acceptable carrier. The invention also relates to a method for the prevention or treatment of autoimmune diseases comprising administering an antibody or antigen-binding fragment thereof, or antibody-drug conjugate thereof, according to the invention in an effective amount necessary for a patient.

Since the composition uses the above-described antibody or antigen-binding fragment thereof specifically binding to IL-17A according to the present invention, the common content between the two is to avoid excessive complexity of the present specification according to the repetitive description. For the sake of brevity, the description is omitted.

The autoimmune diseases include rheumatoid arthritis, psoriasis, dry arthritis, ankylosing spondylitis, multiple sclerosis, interstitial fibrosis, lupus, glomerulonephritis, Crohn's disease, inflammatory bowel disease, autoimmune eye disease, arthritis of children, Benze disease, IL-1 Deficiency of receptor antagonist (DIRA), TNF receptor related periodic syndrome (TRAPS), neonatal multiple inflammatory disease (NOMID), familial thalamic fever (FMF), and cryopyrin related periodic syndrome (CAPS) However, the present invention is not limited thereto.

The present invention comprises, for example, (a) a pharmaceutically effective amount of an antibody or antigen-binding fragment thereof according to the invention, or said antibody-drug conjugate; And (b) it may be a pharmaceutical composition for the prevention or treatment of cancer comprising a pharmaceutically acceptable carrier. The invention also relates to a method for the prevention or treatment of cancer comprising administering to a patient an effective amount necessary for an antibody or an antigen-binding fragment thereof, or said antibody-drug conjugate.

The cancer may be, for example, melanoma, lung cancer, liver cancer, glioblastoma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, renal cell cancer, gastric cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, non-Hodgkin's lymphoma, Hodgkin's May be one or more selected from the group consisting of lymphoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, isolated myeloma and aplastic anemia It is not limited.

"Prevention" means any action that inhibits or delays the progression of autoimmune disease or cancer with administration of a composition according to the present invention, and "treatment" means the inhibition of autoimmune disease or cancer development, autoimmune disease or cancer It means relief or cure.

Pharmaceutically acceptable carriers included in the compositions of the present invention are those commonly used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. The composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, pulmonary administration and rectal administration Or the like.

In oral administration, because proteins or peptides are digested, oral compositions should be formulated to coat the active agent or to protect it from degradation in the stomach. In addition, the pharmaceutical composition may be administered by any device in which the active agent may migrate to the target cell.

Suitable dosages of the compositions according to the invention vary depending on factors such as the method of formulation, mode of administration, age, weight, sex, morbidity, condition of the patient, food, time of administration, route of administration, rate of excretion and reaction sensitivity, The skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment or prophylaxis. For example, the daily dose of the pharmaceutical composition of the present invention may be administered at a dose of 0.0001-100 mg / kg, for example 1 mg / kg to 2 g / kg. The antibody or antigen-binding fragment thereof can be administered single or multiple times. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to prevent or treat autoimmune disease.

The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents.

The compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient, according to methods which can be easily carried out by those skilled in the art. It can be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of extracts, powders, suppositories, powders, granules, tablets, or capsules, and may further include a dispersant or stabilizer.

EXAMPLE

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1: IL-17 Antigen Expression and Purification

1. Construction of IL-17A Protein Expression Vector

Cloning of IL-17A was based on human IL-17A cDNA (sinobiological, China: SEQ ID NO: 58), using primers for IL-17A at 5 'and 3' (Table 1) to obtain only the extracellular domain. Amplification was carried out by polymerase chain reaction (PCR). The amplified PCR product was prepared by fusing His-tag to the carboxy-terminus using N293F vector (FIG. 1). IL-17A-N293F was transformed into DH5α Escherichia coli cells to select ampicillin resistant strains. The cleavage and sequencing by sfiI and xhoI restriction enzymes confirmed the insertion of IL-17A-His gene (SEQ ID NO: 59).

TABLE 1

2. Expression and Purification of IL-17A Antigen

In order to produce human IL-17A-His or mouse IL-17A-His antigen, HEK293F cells were cultured in freestyle media (Gibco, cat. A13835), inoculated at 5 x 10 ⁵ cells / ml, and prepared by incubating daily. IL-17A-N293F vector was transfected into PEI (polyethylenimine, Aldrich, cat. 408727) in HEK293F cells. Plasmid and PEI were mixed at a weight ratio of 1: 2 to form a polyplex, and then transfected into cells. Valproic acid (VPA, valproate, Sigma-P4543) 1 nM was added to the transfected cells, and then cultured for 7 days. Cell cultures cultured for 7 days were transferred to PVDF membrane after SDS-PAGE to treat anti-His HRP antibody and confirmed expression with ECL substrate (FIG. 2A).

The expressed hIL-17A-His and mIL-17A-His cultures were separated and purified by Ni-NTA column, and then stained with CBR-250 by SDS-PAGE to analyze proteins. Glycosylation of the 68th amino acid residue showed bands of various sizes (FIG. 2B).

Example 2: Selection of IL-17 Human Antibodies

1. Antigen Preparation

Blocking was performed after coating 50 ug of the human IL-17A-His protein antigen prepared in Example 1 onto an immunosorbent tube. Bovine serum albumin and skim milk (Becton Dickinson and Company, USA) were used for blocking negative antigens and blocking for nonspecific binding.

2. Preparation of Human Antibody Library

The human antibody library of 2.7 x 10 ¹⁰ variety was infected with 20MOI helper phage and then incubated at 30 ° C for 16 hours to induce expression in scFv-phage form. The cultured bacteria were centrifuged to obtain supernatant, concentrated by treatment with PEG (polyethylene glycol) (Sigma aldrich, USA), and then dissolved in PBS buffer to prepare a human antibody phage library.

3. Bio Panning

The human antibody phage library was reacted with bovine serum albumin-adsorbed immune tube (immune tube) for 37 ° C for 1 hour to induce nonspecific phage antibody binding. Then, only the supernatant was taken and added to the immune tube adsorbed with human IL-17A-His, followed by reaction at 37 ° C. for 2 hours to allow scFv-phage binding to occur. Immune tubes with supernatant removed were washed 5 times with PBS-0.05% Tween 20. 100 μl of 100 mM triethylamine (Sigma aldrich, USA) solution was allowed to react for 10 minutes to allow phages bound to antigen to elute, and the eluted phages were neutralized by 50 μl of 1M Tris pH7.5.

Neutralized phage eluate was added to 10 ml of mid-log XL1-Blue Escherichia coli cell line to induce infection for 1 hour at 37 ° C. Infected XL1-Blue Escherichia coli cell line was spread on agar plate containing Chloramphenicol and cultured at 37˚C for 16 hours to confirm CFU. With the amplified phage in the first panning, only the number of times was increased in the PBST washing step, and the second and third panning were performed in the same manner.

When checking the CFU of the hIL-17A phage antibody pool derived through the 1st to 3rd round panning, it was confirmed that the output CFU of the 3rd panning was increased about 10 times than the output CFU of the 1st to 2nd panning (Table 2). ).

TABLE 2

4. Poly Phage ELISA

Polyphage enzyme linked immunoassay (ELISA) was performed to determine the specificity of the antigen to the positive poly ScFv-phage antibody pool obtained through each round of panning. Direct ELISA to the phage pool obtained in each round on an immuno-plate coated with mouse IL-17A-His antigen to confirm the cross-linking between human IL-17A-His antigen and mouse antigen used for panning. direct ELSIA). As a result, the binding ability to the hIL-17A-His antigen was increased mainly in the tertiary poly ScFv-phage, and it was confirmed that the anti-IL-17A phage antibody was increased in the positive phage pool of the third panning (FIG. 3).

5. Monophage ELISA

Randomly selected E. coli containing single clones from polyphage ELSIA from tertiary panning, which were randomized, were screened at 37 ° C 300 rpm in 96-deep well plates treated with 2xYT / C culture. Helper phage infection was induced after incubation in the mid-log phase. Cells were pelleted by centrifugation at 6000 rpm for 10 minutes to obtain only eluted mono scFv-phage present in the supernatant.

Human IL-17A-His, BSA were coated on a Maxisorb 96well ELISA plate (Nunc, Denmark) at 0.4 μg / ml, respectively, blocked with PBST containing 3% skim milk, and then the obtained mono scFv-phage was digested with each antigen. Transferred to this coated immuno-plate. The ELISA plate was washed three times with 0.05% PBST, and reacted for 1 hour by diluting the anti-M13-HRP (Horse Radish Peroxidase) antibody in PBST 0.05%. The reaction was stopped by developing OPD (o-phenylenediamine dihydrochloride) substrate for 10 minutes and then treated with 2N H ₂ SO ₄ to stop the reaction. The colorated ELISA was measured for absorbance at 490nm using a SpectraMax ELISA reader (Molecular device, USA). As a result, dozens of mono scFv-phage clones were selected, as compared to the signal binding to BSA used as a control antigen, at least three times stronger than the binding signal to hIL-17A-his (FIG. 4).

6. Grouping

Phagemid DNA was isolated using the DNA purification kit (Qiagen, Germany) for the selected clones. Sequences of the isolated DNA, the heavy chain and the light chain were analyzed to identify specific clones with different sequences.

Example 3: Production of Anti-IL-17A Human Antibody

1. Conversion to IgG Form

In order to convert monoclonal phages carrying individual sequences from scFv form to IgG form by sequencing, the nucleotide sequences of the variable regions of the heavy and light chains were cloned into N293F vectors using SfiI / NheI restriction sites.

2. Human Antibody Production

Cloned N293FH and N293FL vectors were co-transfected into HEK293F cells at a 6: 4 ratio. The harvested 7-day culture was centrifuged at 8,000 rpm for 30 minutes to remove cell debris, and the cells were passed through a 0.22 um pore size bottle top filter (Millipore, Steritop-GP Filter Unit. Cat.No. SCGPS01RE). And suspended solids were removed.

3. Purification of Antibodies

(1) Protein A Chromatography

In order to perform the purification, 4 ml of Protein A Sepharose resin slurry (GE Healthcare Life Sciences, Cat. No. 17-1279-04) was added to an empty column (Bio-rad, BR731-1550), followed by 100 ml of DPBS (LB001). The resin was packed and washed with -02). Filtered medium was added to the packed resin, and 1 ml per minute was poured. (Bio-Rad, EP-1 Econo pump, USA) After washing with 150ml DPBS, extracted with 10ml 0.1M glycine-HCl (pH3.3). The extract was neutralized by adding 10% 1M Tris-HCl (pH 9.0) to neutralize the pH, and the buffer was changed to DPBS using Amicon Ultra-15 (Millipore, UFC901096). After this process about 3 times and stopped when concentrated to 1ml, the concentration was checked. Concentration was measured by Nano-drop.

(2) SDS-PAGE

SDS-PAGE was performed to confirm the purity of the purified protein. 3 ug of purified protein sample and 5ul of reducing sample buffer and non-reducing sample buffer were mixed and boiled for 3 min at 100 ° C.

Fill the running tank Bio-Rad (165-8027) with 12% gel, fill the 1x running buffer inside the gel, and fill the outside 1/3 with the pipette to prevent bubbles from entering the prepared sample. The wells were loaded. 4ul of AccuBand Prestained Protein Marker was loaded into the leftmost well. The top cover of the gel chamber was mounted and connected to a power source to run at 200 V and 1 hr.

After one hour the gel was removed from the tank and stained on a 1hr stirrer to the extent of being immersed in staining buffer. Thereafter, the dye solution was discarded and decolorized with a decolorizing solution for about 1 hour. After the decolorized gel was washed with D.W and stored using an image equipment.

(3) Western Blotting

SDS-PAGE was performed to confirm the purity of the purified protein. 3 ug of purified protein sample and 5ul of reducing sample buffer and non-reducing sample buffer were mixed and boiled for 3 min at 100 ° C.

Fill the running tank Bio-Rad (165-8027) with 12% gel, fill the 1x running buffer inside the gel, and fill the outside 1/3 with the pipette to prevent bubbles from entering the prepared sample. The wells were loaded. 4ul of AccuBand Prestained Protein Marker was loaded into the leftmost well. The top cover of the gel chamber was mounted and connected to a power source to run at 200 V and 1 hr.

After an hour, the gel was separated from the tank, the NC membrane was placed under the gel, the cassette was fixed, placed in a transfer tank (Bio-Rad, 165-8027), filled with a transfer buffer, and then run at 110V for about 2 hours.

When expected transfer was complete, the transfer was stopped and the NC membrane was taken out and blocked for 1 hr at RT with a blocking solution (1% Skim milk). Then, the secondary antibody (Fc-HRP (Thermo, 31413), His-HRP (SIGMA, A7058)) 1: 4000 was added to the blocking solution and incubated for 1hr at RT. Washed 5 times 10ml each with 1xPBST. The ECL solution (Intron, 16024) was then scattered on the NC membrane and detected by Chemi Doc (UVITEC, mini HD). The sequences of the obtained antibodies are as described in Tables 3 and 4.

TABLE 3 Heavy chain CDRs and heavy chain variable regions

TABLE 4 Light Chain CDRs and Light Chain Variable Regions

EXAMPLE  4: anti-IL-17A of human antibody Neutralization  analysis

1. IL-17 ligand-receptor binding inhibition assay

Candidates converted to IgG form were analyzed by competitive ELISA to inhibit hIL-17A and hIL-17 receptor binding. This experiment can directly identify whether Ligand-Receptor binding is inhibited by candidate antibodies, which can primarily explain the mechanism of efficacy results in cell-based experiments and animal model experiments. have. In addition, there is also a meaning as a selection technique that can be selected with a high probability of candidates that can be effective in the IL-17A neutralization experiment of the cell-based experiment. Human IL-17 RA-Fc (R & D system, USA) was blocked with 3% skim milk PBST by O / N coating on ELISA plate (Nunc, USA) at 1 ug / ml. Human IL-17A-His prepared as in Example 1 was diluted 1 ug / ml and mixed without antibody candidates or antibody candidates diluted with 666 nM, 66 nM, and 6 nM and reacted at room temperature for 1 hour. The mixture was treated in a blocked ELISA plate and allowed to stand at 37 ° C. for 1 hour, followed by treatment with an anti-His-HRP antibody diluted 1: 3000. Tetramethylbenzidine (Sigma aldrich, USA) substrate was treated with 10 minutes of color development and then treated with 2N H2SO4 to stop the reaction. The developed ELISA was measured for absorbance at 450nm using a SpectraMax ELISA reader (Molecular device, USA). Nucleotide and amino acid sequences of secukinumab, a positive control, were prepared with reference to US Patent No. 7805155, and the sequences of the light and heavy chains were cloned into N293F, our high expression temporary vector, into 40 mL of HEK293F cells. Seckinumab was purified from the cultures after transfection and incubation for 6 days.

Experimental results show that clones such as 7D1, 3G9, 4D3, 9E12 and the like inhibit the ligand-receptor binding, and 7D1 shows a binding inhibitory effect close to the control group secukinumab (FIG. 5). .

The competition ELISA was repeated for the top 6 candidates showing binding inhibition including 7D1. Each candidate antibody was diluted 3 fold at 100 ug / ml to 0.0457 ug / ml and mixed with human IL-17A-His. As a result, it was confirmed that the binding inhibitory ability in the order of 7D1> 9E12> 3G9> 4D3 ≒ 6C2 ≒ 9E5 (FIG. 6).

2. Anti-IL-17A Candidate Neutralizing Analysis by Cell-Based Efficacy

Antibody candidates showing Ligand-Receptor binding inhibition were evaluated for their ability to induce binding inhibition and neutralize hIL-17A in cell-based experiments. HeLa (Homo sapiens cervix adenocarcinoma) (ATCC, USA), known to secrete the inflammatory cytokine hIL-6 due to IL-17A, was seeded in 96-well cell culture plates by 2x10 ⁴ cells at 37 ° C, 5 % CO _{2 was} incubated for at least 16 hours. Then, each antibody diluted with 10ng / ml of hTNF-α (R & D system, USA), 50ng / ml of hIL-17A (R & D system, USA), and 10ug / ml was mixed and reacted at 37 ° C for 1 hour. The mixture was treated with HeLa cell line and incubated together for 24 hours. After that, only the culture medium was taken and treated in the IL-6 measurement Duoset ELISA kit (R & D system, USA), and the degree of neutralization of the antibody was measured using a spectrophotometer (SpectraMax M5 spectraphotometer, Molecular Devices, USA).

It was confirmed that the candidate antibody group was able to inhibit IL-6 secretion level of the TNF-α alone treatment group by targeting only IL-17. In case of 7D1 and 9E12 which have ligand-receptor binding inhibitory ability through competitive ELISA, the neutralizing ability was the highest in cell-based efficacy evaluation, while 9E5 showed no cell-based inhibitory activity in competitive ELISA. Efficacy evaluation showed a neutralizing capacity similar to 9E12 (FIG. 7).

Example 5 Alteration of the Heavy Chain Chain for Promoting Affinity

1. Optimization of Complementarity Determination Sites

Optimized libraries were constructed to enhance antigen specific affinity for selected anti-IL-17A antibody clones 7D1, 9E12. The construction of the optimization library was attempted to intensively improve the complementarity determining region (CDR) in the variable region of the heavy chain. Polymerase chain reaction (PCR) using heavy chain chain variable regions of 7D1 and 9E12 as a template, using primers set up to induce mutations in Solg PFU-X (solgent, Korea) and CDR2 or CDR3 regions (Table 5). To obtain a PCR product containing a variant of the heavy chain CDR region of each antibody. PCR products were separated by electrophoresis on 1% agarose gel and purified by QG buffer (Qiagen, Germany). Each PCR product was overlapped to make one PCR product and inserted into a phagemid vector (FIG. 8). Phagemid vector (phagemid vector) was transformed into XL1-Blue cells, plated in 2xYT / C agar and incubated for 15 hours at 37 ℃. Complementary region variant libraries prepared with 7D1 and 9E12 templates were made with a size of 9.03 × 10 ⁵ cfu, 5.91 × 10 ⁵ cfu.

In order to select an antibody having an improved affinity of IL-17A, phage display and panning were performed up to the third round panning as in Example 2, and the intensity of the wash step was adjusted to induce positive phage. As in the manner described in Example 2, polyclonal phage and monoclonal phage were analyzed to select single clones of scFv-phage and converted to IgG form.

TABLE 5

Competitive ELISA was performed as in Example 4 to select clones with better binding inhibitory ability than the parent antibodies 7D1 and 9E12 in the IgG form. In this experiment, since parental antibody 7D1 had better binding inhibitory activity than parental antibody 9E12, 9E12 was excluded from this analysis. 7H3C11, 7H3B6, 7H3C12, 9H2D9 derived through alteration of complementarity determining sites showed IC ₅₀ results improved over the binding inhibition of parental antibody 7D1 (FIG. 9). Of these, 7H3B6 IC ₅₀ = 34.63nM, 9H2D9 IC ₅₀ = 34.63nM four antibodies showed improved binding inhibition than the parental antibody 7D1 (Table 6).

TABLE 6

2. Optimization of structure formation site

Of the four antibody candidates, three candidates 7H3C11, 7H3B6, and 7H3C12 were subjected to germlining of the structure forming site (framework region: Framework region). The technique enhances homology by comparing the amino acid sequence of the antibody to be selected with the human germline amino acid sequence, thereby increasing the immunogenicity, ADA (immunogenicity) that can occur when treated with a patient. This is to prevent the anti-drug-antibody and to maintain the binding ability of the parent antibody. IGBLAST (NCBI, USA) was used to compare the homology of the structure formation sites, and the structure formation site was changed by cloning using the germline having the highest homology as a template. The modified 7H3C11FW, 7H3B6FW, 7H3C12FW and 9H2D9 were produced and purified as in Example 3 and the purity was analyzed using SDS-PAGE (FIG. 10).

The 7H3C11FW, 7H3B6FW, and 7H3C12FW, which induced mutations in the structure-forming site from 7H3C11, 7H3B6, and 7H3C12 with enhanced affinity complementarity, have a melting point of 1 ° C in a thermal shift assay conducted under temperature harsh conditions. 66 ° C. → 67 ° C.), productivity increased by 55% or decreased by 18% (Table 7).

TABLE 7

3. Binding Specificity Analysis of Optimized Clones

The CDR region, which is known as an important region of antigen antibody binding, is the position where the variation and diversity of the sequence is the largest among regions constituting the antibody. CDR maturation may result in enhancement of target binding capacity, improvement of neutralizing effect, but rather loss of binding power or nonspecific binding power may occur. Accordingly, the binding capacity of the CDR maturated clone was reanalyzed.

ELISA was performed to confirm antigen specific binding specificity of the optimized clones. Human IL-17A and Mouse IL-17A (R & D system, USA), marmoset IL-17A (Sino biological, China), cynomolgous IL-17A, human IL-17F, human IL-17A / F (R & D system, USA), BSA was coated with O / N at 37 ° C. in 96 well plates at 15 nM each. After blocking the uncoated portion with 3% skim milk / PBST 0.05%, 66 nM, 6.6 nM, 0.6 nM IL-17A antibody was added to the plate and reacted at 37 ° C. for 1 hour, and anti-human Fc- HRP (pierce, USA) was diluted 1: 3000 and reacted for 1 hour. Next, the TMB substrate (BD, USA) was treated for 10 minutes to develop the color, followed by 2N H ₂ SO ₄ treatment to stop the reaction. The colorated ELISA was measured for absorbance at 450nm using Sunrise ELISA reader (TECAN, Switzerland).

The results are shown in FIGS. 11A and 11B, and with reference to FIG. 11A, 7H3C11FW, 7H3B6FW, 7H3C12FW, 9H2D9, Secukinumab are human, marmoset, cynomolgus species and human IL-17A / except mouse IL-17A. It maintains binding to the F heterodimer. IL-17B, IL-17C, IL-17D, IL-17E and human TNFα, human RAGE-his, human ErbB2-his, human VEGF-his corresponding to IL-17 ligand family as shown in FIGS. 11B and 11C No binding to, non-target antigen proteins of human CD19-His, human HMGB1-His was shown. Characteristic, it was confirmed that there is no binding ability to human IL-17F, unlike the control, cecukinumab. According to the EMA Assessment report (EMA / CHMP / 389874/2014), Secukinumab has been reported to have weak binding capacity to human IL-17F, which is shown to be identical to the present results. The antibodies of the invention proved to be very specific antibodies with enhanced binding to IL-17A only.

4. Neutralization Analysis of Maturated Clones

The neutralization capacity of four optimized clones whose binding characteristics were identified were measured. Inhibitory concentration 50 (IC ₅₀ ) at which 50% of the IL-17A inducible response was inhibited by the antibody was measured using HeLa cells. HeLa (Homo sapiens cervix adenocarcinoma) (ATCC, USA), known to secrete the inflammatory cytokine hIL-6 due to IL-17A, was seeded in 96-well cell culture plates by 2x10 ⁴ cells at 37 ° C, 5 % CO _{2 was} incubated for at least 16 hours. Subsequently, 200mM of hTNF-α (R & D system, USA) was treated with 3.3nM of hIL-17A (R & D system, USA) to stimulate IL-6 expression and anti-IL-17A antibody was treated from 177nM to 80pM. Diluted three times until treatment was 37 ℃, 1 hour reaction. The mixture was treated with HeLa cell line and incubated together for 24 hours. Thereafter, only the culture medium was taken and treated in the IL-6 measurement Duoset ELISA kit (R & D system, USA), and the degree of neutralization of the antibody was measured using a spectrophotometer (SpectraMax M5 spectraphotometer, Molecular Devices, USA).

Antibodies of the invention demonstrated that concentration-dependent inhibition of IL-6 secreted IL-17A dependent. 9H2D9 is IC ₅₀ = 2.38nM, which is about 1.7 times better IC ₅₀ than the positive control secukinumab in-house IC ₅₀ = 4.16nM, and 7H3C11 FW is IC ₅₀ = 2.88nM, which is 1.4 times IC _50. Excellent. The negative control hIgG (Sigma Aldrich, USA) did not inhibit IL-6 secretion. Table 8 and FIG. 12.

The 7H3C11 FW and 9H2D9 clones, which exhibited superior inhibition of Ligand-Receptor binding through competitive ELISA, were also shown to be superior in cell-based IC ₅₀ results. However, in the case of the 7H3B6FW clone, the results of the two experiments were inconsistent, which resulted in a slight decrease in binding capacity during germlining.

The anti-IL-17A antibody of the present invention is a very specific antibody that binds only to the IL-17A antigen, compared to the existing anti-IL-17A antibody, and induces neutralization of IL-17A to effectively inhibit inflammatory cytokine secretion. .

TABLE 8

As described above in detail a specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Y-Biologics Inc. <120> Antibodies Specifically Binding Against IL-17A and Uses Thereof <130> P16-B333 <160> 59 <170> KopatentIn 2.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> H-CDR1 <400> 1 Ser Tyr Thr Met His   1 5 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> H-CDR2 <400> 2 Ile Ser Phe Asp Gly Arg Ser Lys Leu Tyr Gly Asp Ser Val Arg Asp   1 5 10 15 <210> 3 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 3 Arg Gly Arg Glu Gly Glu Asp Ala Phe Asp Leu   1 5 10 <210> 4 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> H-CDR2 <400> 4 Ile Ser Phe Asp Gly Arg Ser Lys Leu Tyr Gly Asp Ser Val Lys Gly   1 5 10 15 <210> 5 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 5 Gly Ser Val Arg Gly Glu Ala Ala Phe Asp Leu   1 5 10 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 6 Gly Ser Lys Leu Gly Glu Asp Ala Phe Asp Leu   1 5 10 <210> 7 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 7 Gly Ser Arg Ile Gly Glu Asp Ala Phe Asp Leu   1 5 10 <210> 8 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> H-CDR1 <400> 8 Asp His Ala Met His   1 5 <210> 9 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> H-CDR2 <400> 9 Ser Leu Ile Ser Gly Asp Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val   1 5 10 15 Lys gly         <210> 10 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 10 His Phe Ser Asp Ser Arg Gly Arg Ser Asp Val Pro Phe Asp Ile   1 5 10 15 <210> 11 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> H-CDR2 <400> 11 Gly Leu Ile Gly Pro Asp Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val   1 5 10 15 Lys gly         <210> 12 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> L-CDR1 <400> 12 Ser Gly Asp Asn Leu Arg Thr Lys Tyr Val Ser   1 5 10 <210> 13 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> L-CDR2 <400> 13 Gln Asp Thr Arg Arg Pro Ser   1 5 <210> 14 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> L-CDR3 <400> 14 Met Thr Trp Asp Val Asp Thr Thr Ser Met   1 5 10 <210> 15 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 15 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Ala Gln Pro Gly Arg   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Gly              20 25 30 <210> 16 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 16 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Thr Leu   1 5 10 15 <210> 17 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 17 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Met Leu Tyr Leu Lys   1 5 10 15 Ile Ser Asp Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg              20 25 30 <210> 18 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> FR4 <400> 18 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser   1 5 10 <210> 19 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 19 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Gly              20 25 30 <210> 20 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 20 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr Leu Gln   1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg              20 25 30 <210> 21 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> FR4 <400> 21 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser   1 5 10 <210> 22 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 22 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp              20 25 30 <210> 23 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 23 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val   1 5 10 <210> 24 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 24 Arg Phe Ile Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr Leu Gln   1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg              20 25 30 <210> 25 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> FR4 <400> 25 Trp Gly Gln Gly Thr Leu Ile Thr Val Ser Ser   1 5 10 <210> 26 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 26 Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Val   1 5 10 <210> 27 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 27 Ser Tyr Glu Leu Thr Gln Ala Pro Ser Leu Ser Val Ser Pro Gly Gln   1 5 10 15 Thr Ala Asn Ile Ile Cys              20 <210> 28 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 28 Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Leu Leu Val Ile Tyr   1 5 10 15 <210> 29 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 29 Gly Ile Pro Ala Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr   1 5 10 15 Leu Thr Ile Ser Gly Thr Gln Thr Arg Asp Glu Ser Thr Tyr Tyr Cys              20 25 30 <210> 30 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> FR4 <400> 30 Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu   1 5 10 <210> 31 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 31 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln   1 5 10 15 Thr Ala Ser Ile Thr Cys              20 <210> 32 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 32 Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr   1 5 10 15 <210> 33 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 33 Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr   1 5 10 15 Leu Thr Ile Ser Gly Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys              20 25 30 <210> 34 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 34 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Ala Gln Pro Gly Arg   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Gly Ser Tyr              20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Thr Leu Ile Ser Phe Asp Gly Arg Ser Lys Leu Tyr Gly Asp Ser Val      50 55 60 Arg Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Met Leu Tyr  65 70 75 80 Leu Lys Ile Ser Asp Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Arg Gly Arg Glu Gly Glu Asp Ala Phe Asp Leu Trp Gly Gln             100 105 110 Gly Thr Met Val Thr Val Ser Ser         115 120 <210> 35 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 35 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Gly Ser Tyr              20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Thr Leu Ile Ser Phe Asp Gly Arg Ser Lys Leu Tyr Gly Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser 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 Ala Arg Gly Ser Val Arg Gly Glu Ala Ala Phe Asp Leu Trp Gly Gln             100 105 110 Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 36 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 36 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Gly Ser Tyr              20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Thr Leu Ile Ser Phe Asp Gly Arg Ser Lys Leu Tyr Gly Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser 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 Ala Arg Gly Ser Lys Leu Gly Glu Asp Ala Phe Asp Leu Trp Gly Gln             100 105 110 Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 37 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 37 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Gly Ser Tyr              20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Thr Leu Ile Ser Phe Asp Gly Arg Ser Lys Leu Tyr Gly Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser 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 Ala Arg Gly Ser Arg Ile Gly Glu Asp Ala Phe Asp Leu Trp Gly Gln             100 105 110 Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 38 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 38 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp His              20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Leu Ile Ser Gly Asp Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser Lys Asn Ser 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 Ala Arg His Phe Ser Asp Ser Arg Gly Arg Ser Asp Val Pro Phe Asp             100 105 110 Ile Trp Gly Gln Gly Thr Leu Ile Thr Val Ser Ser         115 120 <210> 39 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 39 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp His              20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Val          35 40 45 Gly Leu Ile Gly Pro Asp Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser Lys Asn Ser 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 Ala Arg His Phe Ser Asp Ser Arg Gly Arg Ser Asp Val Pro Phe Asp             100 105 110 Ile Trp Gly Gln Gly Thr Leu Ile Thr Val Ser Ser         115 120 <210> 40 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 40 Ser Tyr Glu Leu Thr Gln Ala Pro Ser Leu Ser Val Ser Pro Gly Gln   1 5 10 15 Thr Ala Asn Ile Ile Cys Ser Gly Asp Asn Leu Arg Thr Lys Tyr Val              20 25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Leu Leu Val Ile Tyr          35 40 45 Gln Asp Thr Arg Arg Pro Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser      50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Thr Arg  65 70 75 80 Asp Glu Ser Thr Tyr Tyr Cys Met Thr Trp Asp Val Asp Thr Thr Ser                  85 90 95 Met Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 <210> 41 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 41 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln   1 5 10 15 Thr Ala Ser Ile Thr Cys Ser Gly Asp Asn Leu Arg Thr Lys Tyr Val              20 25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr          35 40 45 Gln Asp Thr Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser      50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met  65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Met Thr Trp Asp Val Asp Thr Thr Ser                  85 90 95 Met Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 <210> 42 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> VH <400> 42 caggtgcagc tggtggagtc tgggggaggc gtggcccagc ctgggaggtc cctcagactc 60 tcctgtgcag cctctggatt cgccttcggt agttacacta tgcactgggt ccgccaggcg 120 ccaggcaagg gactggagtg ggtgacactt atatcgtttg atggacgtag caagctttac 180 ggagactccg tgagggaccg attcaccatc tccagagaca attccaagaa catgctgtat 240 ctgaaaataa gtgacctgcg atctgaggac acggccgtgt attactgtgc gagacggggg 300 agggagggtg aagatgcttt cgatctctgg ggccaaggga caatggtcac cgtctcctca 360                                                                          360 <210> 43 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> VH <400> 43 caggtgcagc tggtggagtc tgggggaggc gtggtgcagc ctgggaggtc cctcagactc 60 tcctgtgcag cctctggatt cgccttcggt agttacacta tgcactgggt ccgccaggcg 120 ccaggcaagg gactggagtg ggtgacactt atatcgtttg atggacgtag caagctttac 180 ggagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cagcctgtat 240 ctgcagatga acagcctgcg agccgaggac acggccgtgt attactgtgc gagagggtct 300 gtgcggggtg aagctgcttt cgatctctgg ggccaaggga cactggtcac cgtctcctca 360                                                                          360 <210> 44 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> VH <400> 44 caggtgcagc tggtggagtc tgggggaggc gtggtgcagc ctgggaggtc cctcagactc 60 tcctgtgcag cctctggatt cgccttcggt agttacacta tgcactgggt ccgccaggcg 120 ccaggcaagg gactggagtg ggtgacactt atatcgtttg atggacgtag caagctttac 180 ggagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cagcctgtat 240 ctgcagatga acagcctgcg agccgaggac acggccgtgt attactgtgc gagagggagt 300 aagttgggtg aagatgcttt cgatctctgg ggccaaggga cactggtcac cgtctcctca 360                                                                          360 <210> 45 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> VH <400> 45 caggtgcagc tggtggagtc tgggggaggc gtggtgcagc ctgggaggtc cctcagactc 60 tcctgtgcag cctctggatt cgccttcggt agttacacta tgcactgggt ccgccaggcg 120 ccaggcaagg gactggagtg ggtgacactt atatcgtttg atggacgtag caagctttac 180 ggagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cagcctgtat 240 ctgcagatga acagcctgcg agccgaggac acggccgtgt attactgtgc gagaggttcg 300 cgtattggtg aagatgcttt cgatctctgg ggccaaggga cactggtcac cgtctcctca 360                                                                          360 <210> 46 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> VH <400> 46 caggtgcagc tggtggagtc tgggggaggc gtggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cgtctggatt cacctttgat gatcatgcca tgcactgggt ccgtcaagct 120 ccagggaagg gtctggagtg ggtctctctt attagcggtg atggtggtgc cacatactat 180 gcagactctg tgaagggccg gttcatcatc tccagagaca acagcaaaaa ctccctgtat 240 ctgcaaatga acagtctgag agccgaggac acggccgtgt attactgtgc gagacatttt 300 tctgatagtc gtggtcgctc cgatgttcct tttgatatct ggggccaagg gacactgatc 360 accgtctcct ca 372 <210> 47 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> VH <400> 47 caggtgcagc tggtggagtc tgggggaggc gtggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cgtctggatt cacctttgat gatcatgcca tgcactgggt ccgtcaagct 120 ccagggaatg gtctggagtg ggtcggcctg attggtcctg atggtggtgc cacatactat 180 gcagactctg tgaagggccg gttcatcatc tccagagaca acagcaaaaa ctccctgtat 240 ctgcaaatga acagtctgag agccgaggac acggccgtgt attactgtgc gagacatttt 300 tctgatagtc gtggtcgctc cgatgttcct tttgatatct ggggccaagg gacactgatc 360 accgtctcct ca 372 <210> 48 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> VL <400> 48 tcctatgagc tgacacaggc accctcactg tccgtgtcgc caggacagac agccaacatc 60 atctgctctg gagataactt gcgtactaaa tatgtttctt ggtatcagca gaagccaggc 120 cagtcccctt tattggtcat ctatcaggac accaggcggc cctcaggcat ccctgcgcga 180 ttctcaggct ccaactcggg gaacacagcc actctgacca tcagcgggac ccagactaga 240 gatgaatcta cctattactg tatgacgtgg gacgtcgaca ctacctcgat gattttcggc 300 ggagggacca agctgaccgt ccta 324 <210> 49 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> VL <400> 49 tcctatgagc tgacacagcc cccctcagtg tccgtgtcgc caggacagac agccagcatc 60 acctgctctg gagataactt gcgtactaaa tatgtttctt ggtatcagca gaagccaggc 120 cagtcccctg tgttggtcat ctatcaggac accaggcggc cctcaggcat ccctgagcga 180 ttctcaggct ccaactcggg gaacacagcc actctgacca tcagcgggac ccaggctatg 240 gatgaagctg actattactg tatgacgtgg gacgtcgaca ctacctcgat gattttcggc 300 ggagggacca agctgaccgt ccta 324 <210> 50 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 50 ggaatcacaa tcccacgaaa t 21 <210> 51 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 51 ggccacatgg tggacaatcg g 21 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 52 cagctatgac catgattacg 20 <210> 53 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 53 gccgtgtatt actgtgcgag annknnknnk nnkggtgaag atgctttcga tc 52 <210> 54 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 54 tgagggtgcc tgtgtcagc 19 <210> 55 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 55 tctcgcacag taatacacgg c 21 <210> 56 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 56 gatggtggtg ccacatacta t 21 <210> 57 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 57 atagtatgtg gcaccaccat cmnnmnnaat mnngscgacc cactccagac cattc 55 <210> 58 <211> 396 <212> DNA <213> Artificial Sequence <220> <223> hIL-17A <400> 58 ggaatcacaa tcccacgaaa tccaggatgc ccaaattctg aggacaagaa cttcccccgg 60 actgtgatgg tcaacctgaa catccataac cggaatacca ataccaatcc caaaaggtcc 120 tcagattact acaaccgatc cacctcacct tggaatctcc accgcaatga ggaccctgag 180 agatatccct ctgtgatctg ggaggcaaag tgccgccact tgggctgcat caacgctgat 240 gggaacgtgg actaccacat gaactctgtc cccatccagc aagagatcct ggtcctgcgc 300 agggagcctc cacactgccc caactccttc cggctggaga agatactggt gtccgtgggc 360 tgcacctgtg tcaccccgat tgtccaccat gtggcc 396 <210> 59 <211> 420 <212> DNA <213> Artificial Sequence <220> <223> IL-17A-His <400> 59 ggaatcacaa tcccacgaaa tccaggatgc ccaaattctg aggacaagaa cttcccccgg 60 actgtgatgg tcaacctgaa catccataac cggaatacca ataccaatcc caaaaggtcc 120 tcagattact acaaccgatc cacctcacct tggaatctcc accgcaatga ggaccctgag 180 agatatccct ctgtgatctg ggaggcaaag tgccgccact tgggctgcat caacgctgat 240 gggaacgtgg actaccacat gaactctgtc cccatccagc aagagatcct ggtcctgcgc 300 agggagcctc cacactgccc caactccttc cggctggaga agatactggt gtccgtgggc 360 tgcacctgtg tcaccccgat tgtccaccat gtggcccatc atcatcatca tcaccatcac 420                                                                          420

Claims (18)

Heavy chain CDR1 of SEQ ID NO: 1, heavy chain CDR2 of SEQ ID NO: 2 and heavy chain CDR3 of SEQ ID NO: 3, and light chain CDR1 of SEQ ID NO: 12, light chain CDR2 of SEQ ID NO: 13, and light chain CDR3 of SEQ ID NO: 14;
Heavy chain CDR1 of SEQ ID NO: 1, heavy chain CDR2 of SEQ ID NO: 4 and heavy chain CDR3 of SEQ ID NO: 5, and light chain CDR1 of SEQ ID NO: 12, light chain CDR2 of SEQ ID NO: 13, and light chain CDR3 of SEQ ID NO: 14;
Heavy chain CDR1 of SEQ ID NO: 1, heavy chain CDR2 of SEQ ID NO: 4 and heavy chain CDR3 of SEQ ID NO: 6, light chain CDR1 of SEQ ID NO: 12, light chain CDR2 of SEQ ID NO: 13, and light chain CDR3 of SEQ ID NO: 14;
Heavy chain CDR1 of SEQ ID NO: 1, heavy chain CDR2 of SEQ ID NO: 4 and heavy chain CDR3 of SEQ ID NO: 7, and light chain CDR1 of SEQ ID NO: 12, light chain CDR2 of SEQ ID NO: 13, and light chain CDR3 of SEQ ID NO: 14;
Heavy chain CDR1 of SEQ ID NO: 8, heavy chain CDR2 of SEQ ID NO: 9 and heavy chain CDR3 of SEQ ID NO: 10, and light chain CDR1 of SEQ ID NO: 12, light chain CDR2 of SEQ ID NO: 13, and light chain CDR3 of SEQ ID NO: 14; or
Specific for IL-17A comprising heavy chain CDR1 of SEQ ID NO: 8, heavy chain CDR2 of SEQ ID NO: 11 and heavy chain CDR3 of SEQ ID NO: 10, and light chain CDR1 of SEQ ID NO: 12, light chain CDR2 of SEQ ID NO: 13, and light chain CDR3 of SEQ ID NO: 14 An antibody or antigen-binding fragment thereof that binds to an enemy.
delete The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region framework (FR) selected from the group consisting of SEQ ID NOs: 15-26. The compound of claim 3, further comprising: FR1 of SEQ ID NO: 15, FR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and FR4 of SEQ ID NO: 18;
FR1 of SEQ ID NO: 19, FR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 20, and FR4 of SEQ ID NO: 21;
FR1 of SEQ ID NO: 22, FR2 of SEQ ID NO: 23, FR3 of SEQ ID NO: 24, and FR4 of SEQ ID NO: 25; And
An antibody or antigen-binding fragment thereof comprising a heavy chain variable region framework selected from the group consisting of FR1 of SEQ ID NO: 22, FR2 of SEQ ID NO: 26, FR3 of SEQ ID NO: 24, and FR4 of SEQ ID NO: 25. The antibody or antigen-binding fragment thereof of claim 1, comprising a light chain variable region framework (FR) selected from the group consisting of SEQ ID NOs: 27-33. The compound of claim 5, further comprising FR1 of SEQ ID NO: 27, FR2 of SEQ ID NO: 28, FR3 of SEQ ID NO: 29, and FR4 of SEQ ID NO: 30; or
An antibody or antigen-binding fragment thereof, comprising a light chain variable region framework of FR1 of SEQ ID NO: 31, FR2 of SEQ ID NO: 32, FR3 of SEQ ID NO: 33, and FR4 of SEQ ID NO: 30. The heavy chain variable region of claim 1, selected from the group consisting of SEQ ID NO: 34 to SEQ ID NO: 39, or
An antibody or antigen-binding fragment thereof, comprising a light chain variable region of SEQ ID NO: 40 or SEQ ID NO: 41. A polynucleotide encoding the antibody according to any one of claims 1, 3 to 7, or an antigen binding fragment thereof. The polynucleotide of claim 8, wherein the polynucleotide is selected from the group consisting of SEQ ID NO: 42 to SEQ ID NO: 49. A vector comprising the polynucleotide of claim 8. A cell transformed with the vector of claim 10. A method of preparing an antibody or antigen-binding fragment thereof that specifically binds IL-17A, comprising the following steps:
(a) culturing the cells of claim 11; And
(b) recovering the antibody or antigen-binding fragment thereof from the cultured cells. A composition for preventing or treating autoimmune diseases comprising the antibody or antigen-binding fragment thereof according to any one of claims 1, 3 to 7 as an active ingredient. The method of claim 13, wherein the autoimmune disease is rheumatoid arthritis, psoriasis, dry arthritis, ankylosing spondylitis, multiple sclerosis, interstitial fibrosis, lupus, glomerulonephritis, Crohn's disease, inflammatory bowel disease, autoimmune ocular disease, child arthritis, Bengal disease, deficiency of IL-1 receptor antagonist (DIRA), TNF receptor-related periodic syndrome (TRAPS), neonatal multiple inflammatory disease (NOMID), familial hypothalamic fever (FMF), and cryopyrin-related periodic syndrome (CAPS) Compositions selected from the group consisting of. A composition for preventing or treating cancer, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 and 3 to 7 as an active ingredient. The method of claim 15, wherein the cancer is melanoma, lung cancer, liver cancer, glioblastoma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, renal cell cancer, gastric cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, isolated myeloma and aplastic anemia Composition. An antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1, 3-7, and a drug bound thereto. A double antibody comprising the antibody of claim 1, or an antigen binding fragment thereof.

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