Specific binding molecules for LRIG-1 proteins and uses thereof

文档序号：1539154 发布日期：2020-02-14 浏览：16次中文

阅读说明：本技术 Lrig-1蛋白的特异性结合分子及其用途 (Specific binding molecules for LRIG-1 proteins and uses thereof ) 是由金廷镐金范锡于 2018-04-18 设计创作，主要内容包括：本发明涉及能够与Lrig-1蛋白特异性结合的结合分子,该蛋白位于调节性T细胞的表面。本发明提供的结合分子可以激活调节性T细胞的功能,以有效预防、改善或治疗由各种免疫细胞和炎性细胞的过度激活或表达引起的疾病,例如免疫相关疾病,如自身免疫疾病、移植物抗宿主病、器官移植排斥、哮喘、特应性疾病、急性或慢性炎性疾病等。此外,本发明的Lrig-1蛋白的特异性结合分子,优选抗体具有以下优点：与先前可商品化购得的抗Lrig-1抗体相比,更有效地靶向Lrig-1蛋白,并且具有非常好的结合能力。(The present invention relates to binding molecules capable of specifically binding to the Lrig-1 protein, which is located on the surface of regulatory T cells. The binding molecules provided by the present invention can activate the function of regulatory T cells to effectively prevent, ameliorate or treat diseases caused by over-activation or expression of various immune cells and inflammatory cells, for example, immune-related diseases such as autoimmune diseases, graft-versus-host disease, organ transplant rejection, asthma, atopic diseases, acute or chronic inflammatory diseases, and the like. Furthermore, specific binding molecules, preferably antibodies, of the Lrig-1 protein of the invention have the following advantages: lrig-1 protein is targeted more efficiently and with very good binding capacity than the anti-Lrig-1 antibodies previously commercially available.)

1. A binding molecule that specifically binds to leucine rich and immunoglobulin-like domain 1(Lrig-1) proteins.

2. The binding molecule of claim 1, wherein said Lrig-1 protein consists of the amino acid sequence set forth in SEQ ID No. 1 or 3.

3. The binding molecule of claim 1, wherein said Lrig-1 protein is encoded by a polynucleotide as set forth in SEQ ID No. 2 or 4.

4. The binding molecule of claim 1, wherein said binding molecule comprises the following:

a heavy chain variable region comprising a heavy chain CDR1 consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs 5, 13, 21, or 29; a heavy chain CDR2 consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs 6, 14, 22, and 30; a heavy chain CDR3 consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs 7, 15, 23, and 31; and

a light chain variable region comprising a light chain CDR1 consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs 8, 16, 24, or 32; a light chain CDR2 consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs 9, 17, 25, and 33; a light chain CDR3 consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs 10, 18, 26, and 34.

5. The binding molecule of claim 1, wherein said binding molecule comprises:

a heavy chain variable region selected from the group consisting of:

(a) a heavy chain variable region comprising heavy chain CDR1 of SEQ ID NO. 5, heavy chain CDR2 of SEQ ID NO. 6 and heavy chain CDR3 of SEQ ID NO. 7;

(b) a heavy chain variable region comprising heavy chain CDR1 of SEQ ID NO. 13, heavy chain CDR2 of SEQ ID NO. 14 and heavy chain CDR3 of SEQ ID NO. 15;

(c) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO:21, the heavy chain CDR2 of SEQ ID NO:22 and the heavy chain CDR3 of SEQ ID NO: 23; and

(d) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO:29, the heavy chain CDR2 of SEQ ID NO:30 and the heavy chain CDR3 of SEQ ID NO: 31;

and

a light chain variable region selected from the group consisting of:

(e) a light chain variable region comprising light chain CDR1 of SEQ ID NO. 8, light chain CDR2 of SEQ ID NO. 9, and light chain CDR3 of SEQ ID NO. 10;

(f) a light chain variable region comprising light chain CDR1 of SEQ ID NO 16, light chain CDR2 of SEQ ID NO 17 and light chain CDR3 of SEQ ID NO 18;

(g) a light chain variable region comprising light chain CDR1 of SEQ ID NO. 24, light chain CDR2 of SEQ ID NO. 25 and light chain CDR3 of SEQ ID NO. 26;

(h) the light chain variable region comprising light chain CDR1 of SEQ ID NO:32, light chain CDR2 of SEQ ID NO:33 and light chain CDR3 of SEQ ID NO: 34.

6. The binding molecule of claim 1, wherein said binding molecule is selected from the group consisting of (1) - (4) below:

(1) a binding molecule comprising a heavy chain variable region comprising heavy chain CDR1 of SEQ ID No. 5, CDR2 of SEQ ID No. 6 and CDR3 of SEQ ID No. 7 and a light chain variable region comprising light chain CDR1 of SEQ ID No. 8, CDR2 of SEQ ID No. 9 and CDR3 of SEQ ID No. 10;

(2) a binding molecule comprising a heavy chain variable region comprising heavy chain CDR1 of SEQ ID No. 13, CDR2 of SEQ ID No. 14 and CDR3 of SEQ ID No. 15 and a light chain variable region comprising light chain CDR1 of SEQ ID No. 16, CDR2 of SEQ ID No. 17 and CDR3 of SEQ ID No. 18;

(3) a binding molecule comprising a heavy chain variable region comprising heavy chain CDR1 of SEQ ID No. 21, CDR2 of SEQ ID No. 22 and CDR3 of SEQ ID No. 23 and a light chain variable region comprising light chain CDR1 of SEQ ID No. 24, CDR2 of SEQ ID No. 25 and CDR3 of SEQ ID No. 26;

(4) a binding molecule comprising a heavy chain variable region comprising heavy chain CDR1 of SEQ ID NO. 29, heavy chain CDR2 of SEQ ID NO. 30 and heavy chain CDR3 of SEQ ID NO. 31 and a light chain variable region comprising light chain CDR1 of SEQ ID NO. 32, light chain CDR2 of SEQ ID NO. 33 and light chain CDR3 of SEQ ID NO. 34.

7. The binding molecule of claim 1, wherein said binding molecule comprises:

a heavy chain variable region consisting of any one amino acid sequence selected from the group consisting of: 11, 19, 27 and 35; and

a light chain variable region consisting of any one of the amino acid sequences selected from the group consisting of: 12, 20, 28 and 36 SEQ ID NOs.

8. The binding molecule of claim 1, wherein said binding molecule is selected from the group consisting of:

a binding molecule comprising the heavy chain variable region of SEQ ID NO 11 and the light chain variable region of SEQ ID NO 12;

a binding molecule comprising the heavy chain variable region of SEQ ID NO 19 and the light chain variable region of SEQ ID NO 20;

a binding molecule comprising the heavy chain variable region of SEQ ID NO. 27 and the light chain variable region of SEQ ID NO. 28; and

a binding molecule comprising the heavy chain variable region of SEQ ID No. 35 and the light chain variable region of SEQ ID No. 36.

9. The binding molecule of claim 1, wherein said binding molecule further comprises an Fc region or constant region.

10. The binding molecule of claim 9, wherein said Fc region is that of an IgG1, IgG2, IgG3, or IgG4 antibody, or is a mixed Fc region.

11. The binding molecule of claim 1, further comprising a heavy chain constant region consisting of an amino acid sequence selected from the group consisting of seq id nos: 37, 39, 41, 42, 43 and 44 SEQ ID NOs.

12. The binding molecule of claim 1, further comprising a light chain constant region consisting of the amino acid sequence set forth in SEQ ID NO 38 or 40.

13. The binding molecule of claim 1, wherein said binding molecule further comprises:

a heavy chain constant region consisting of the amino acid sequence shown in SEQ ID NO. 37; and

a light chain constant region consisting of the amino acid sequence shown in SEQ ID NO. 38.

14. The binding molecule of claim 1, wherein said binding molecule further comprises:

a heavy chain constant region consisting of the amino acid sequence shown in SEQ ID NO 39, 41, 42 or 43; and

a light chain constant region consisting of the amino acid sequence shown in SEQ ID NO. 40.

15. The binding molecule of claim 1, wherein said binding molecule further comprises:

a heavy chain constant region consisting of the amino acid sequence shown in SEQ ID NO. 44.

16. The binding molecule of claim 1, wherein said binding molecule is selected from the group consisting of:

a binding molecule comprising a heavy chain as set forth in SEQ ID NO 45 and a light chain as set forth in SEQ ID NO 46;

a binding molecule comprising a heavy chain as set forth in SEQ ID NO 47 and a light chain as set forth in SEQ ID NO 48;

a binding molecule comprising a heavy chain as set forth in SEQ ID NO. 49 and a light chain as set forth in SEQ ID NO. 50; and

a binding molecule comprising a heavy chain as shown in SEQ ID NO 51 and a light chain as shown in SEQ ID NO 52.

17. The binding molecule of claim 1, wherein said binding molecule is an antibody or fragment thereof.

18. The binding molecule of claim 17, wherein said antibody is a chimeric antibody, a humanized antibody, a bivalent/bispecific molecule, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a diabody, a triabody, or a tetrabody, or a fragment thereof.

19. A nucleic acid molecule encoding the binding molecule of any one of claims 1-18.

20. An expression vector into which the nucleic acid molecule of claim 19 is inserted.

21. A host cell line transfected with the expression vector of claim 20.

22. An antibody-drug conjugate comprising:

an antibody; and

the medicine is a mixture of medicines and a feed additive,

wherein the antibody comprises:

23. A pharmaceutical composition for preventing or treating an immune-related disease, comprising the binding molecule of any one of claims 1 to 18 as an active ingredient.

24. The pharmaceutical composition of claim 23, wherein the immune-related disease is an autoimmune disease, graft versus host disease, organ transplant rejection, asthma, an atopic disease, or an acute or chronic inflammatory disease.

25. A method of preventing or treating an immune-related disorder, comprising:

administering to a subject in need of treatment the binding molecule of any one of claims 1-18, thereby preventing or treating the immune-related disorder.

Technical Field

The present invention relates to binding molecules capable of specifically binding to leucine-rich and immunoglobulin-like domain 1(Lrig-1) proteins, which are proteins present on the surface of regulatory T cells (Treg cells), and uses thereof.

Background

One of the most important features in all normal individuals is the ability to recognize and eliminate non-self antigens without adversely reacting the antigenic materials that make up themselves. Thus, a body's unresponsiveness to self-antigens is referred to as immunological unresponsiveness or tolerance. Self-tolerance occurs by elimination of lymphocytes that may have specific receptors for self-antigens, or by self-inactivation of the ability to respond upon exposure to self-antigens. In cases where problems arise in the induction or maintenance of self-tolerance, an immune response to a self-antigen occurs, and the resulting disease is referred to as an autoimmune disease.

For the treatment of autoimmune diseases, Gershon first proposed the concept of suppressive T cells in the early 70's of the 20 th century, suggesting that there may be T cells that could control and suppress effector functions of conventional T cells (Gershon and k. kondo, Immunology, 1970, 18: 723-37). Since then, studies have been carried out in many fields of immunology to elucidate the biological properties and functions of regulatory T cells.

In this regard, regulatory T cells (Treg cells) have been reported to play an important role in naturally preventing the development of excessive inflammation and immune responses; however, in the case of autoimmune diseases and chronic inflammatory diseases, the function and number of regulatory T cells is significantly reduced. Therefore, in the case of patients with immune and inflammatory diseases, it is important that regulatory T cells are produced at normal levels as one of the treatments for these diseases.

To date, studies have been made on genes and proteins that specifically exist in regulatory T cells, and it has been suggested that substances such as CD25, CTLA4, CD62L, CD38, CD103, GITR, and CD45RB may correspond to marker substances. However, there are no genes and proteins that can only target regulatory T cells alone.

On the other hand, there are three hypervariable regions (hereinafter referred to as "CDRs") called complementarity determining regions and four framework regions. CDRs are primarily used to bind epitopes on antigens. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 in that order from the N-terminus, and are also distinguished by the chain in which the particular CDR is located.

Technical problem

The aim of the invention is to provide specific binding molecules for the Lrig-1 protein present on the surface of regulatory T cells (Treg cells).

It is another object of the invention to provide nucleic acid molecules encoding the binding molecules of the invention.

Another object of the present invention is to provide an expression vector into which the nucleic acid molecule of the present invention is inserted.

It is still another object of the present invention to provide a host cell line transfected with the expression vector of the present invention.

It is still another object of the present invention to provide the antibody-drug conjugate of the present invention.

It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating immune-related diseases, comprising the binding molecule of the present invention.

However, the technical problems to be solved by the present invention are not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood from the following description by those skilled in the art.

Technical scheme for solving problems

The present inventors have found that an Lrig-1 protein exists specifically on the surface of regulatory T cells, selected an epitope on the protein, and produced a monoclonal antibody capable of specifically binding to the Lrig-1 protein, thereby completing the present invention.

One embodiment of the present invention provides binding molecules that specifically bind to leucine rich and immunoglobulin-like domain 1(Lrig-1) proteins.

The term "binding molecule" as used herein refers to a variable domain comprising an intact immunoglobulin, including a monoclonal antibody (e.g., a chimeric, humanized or human monoclonal antibody), or an immunoglobulin that binds an antigen, e.g., an immunoglobulin fragment that competes with the intact immunoglobulin for binding to monomeric HA or trimeric HA of influenza a virus. Regardless of structure, an antigen-binding fragment binds to the same antigen that is recognized by an intact immunoglobulin. The antigen-binding fragment may include a peptide or polypeptide that contains an amino acid sequence of two or more consecutive residues, 20 or more consecutive residues, 25 or more consecutive residues, 30 or more consecutive residues, 35 or more consecutive residues, 40 or more consecutive residues, 50 or more consecutive residues, 60 or more consecutive residues, 70 or more consecutive residues, 80 or more consecutive residues, 90 or more consecutive residues, 100 or more consecutive residues, 125 or more consecutive residues, 150 or more consecutive residues, 175 or more consecutive residues, 200 or more consecutive residues, or 250 or more consecutive amino acid residues in the amino acid sequence of the binding molecule. The term "antigen-binding fragment" specifically includes: fab, F (ab')2, Fv, dAb, Fd, Complementarity Determining Region (CDR) fragments, single chain antibodies (scFv), bivalent single chain antibodies, single chain phage antibodies, diabodies, triabodies, tetrabodies, polypeptides that contain one or more immunoglobulin fragments sufficient for binding a particular antigen to the polypeptide, and the like. The fragments may be produced synthetically, or by enzymatic or chemical digestion of intact immunoglobulins, or may be produced by genetic engineering methods using recombinant DNA techniques. Methods of production are well known in the art.

In the present invention, the Lrig-1 protein is a 1091 amino acid transmembrane protein, consisting of an extracellular or luminal Leucine Rich Repeat (LRR) and three immunoglobulin-like domains, a cellular transmembrane sequence, and a cytoplasmic tail, present on the surface of regulatory T cells. The LRIG gene family includes LRIG1, LRIG2, and LRIG3, and amino acids between them are highly conserved. The LRIG1 gene is highly expressed in normal skin and can be expressed in basal and follicular cells in order to regulate the proliferation of epithelial stem cells. Thus, the LRIG1 gene plays an important role in maintaining epidermal homeostasis, and its deletion may progress to psoriasis or skin cancer. It has been reported that cancer cells may develop when the portion of chromosome 3p14.3 where LRIG1 is located is excised. In fact, it has been determined that expression of LRIG1 is greatly reduced in renal cell carcinoma and cutaneous squamous cell carcinoma. Recently, Lrig-1 has also been found to be expressed only in about 20% to 30% of cancers. On the other hand, for the purposes of the present invention, an Lrig-1 protein may be, but is not limited to, a protein found in humans or mice.

In the present invention, the Lrig-1 protein may be, but is not limited to, a human-derived polypeptide shown in SEQ ID NO. 1 or a mouse-derived polypeptide shown in SEQ ID NO. 3.

In addition, in the present invention, the Lrig-1 protein represented by SEQ ID NO. 1 may be encoded by the polynucleotide represented by SEQ ID NO. 2, but is not limited thereto.

In addition, in the present invention, the Lrig-1 protein represented by SEQ ID NO. 3 may be encoded by the polynucleotide represented by SEQ ID NO. 4, but is not limited thereto.

In the present invention, the binding molecule may be a binding molecule comprising:

a heavy chain variable region selected from the group consisting of:

(a) a heavy chain variable region comprising heavy chain CDR1 of SEQ ID NO. 5, heavy chain CDR2 of SEQ ID NO. 6 and heavy chain CDR3 of SEQ ID NO. 7;

(b) a heavy chain variable region comprising heavy chain CDR1 of SEQ ID NO. 13, heavy chain CDR2 of SEQ ID NO. 14 and heavy chain CDR3 of SEQ ID NO. 15;

(c) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO:21, the heavy chain CDR2 of SEQ ID NO:22 and the heavy chain CDR3 of SEQ ID NO: 23; and

(d) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO:29, the heavy chain CDR2 of SEQ ID NO:30 and the heavy chain CDR3 of SEQ ID NO: 31; and

a light chain variable region selected from the group consisting of:

(e) a light chain variable region comprising light chain CDR1 of SEQ ID NO. 8, light chain CDR2 of SEQ ID NO. 9, and light chain CDR3 of SEQ ID NO. 10;

(f) a light chain variable region comprising light chain CDR1 of SEQ ID NO 16, light chain CDR2 of SEQ ID NO 17 and light chain CDR3 of SEQ ID NO 18;

(g) a light chain variable region comprising light chain CDR1 of SEQ ID NO. 24, light chain CDR2 of SEQ ID NO. 25 and light chain CDR3 of SEQ ID NO. 26; and

(h) the light chain variable region comprising light chain CDR1 of SEQ ID NO:32, light chain CDR2 of SEQ ID NO:33 and light chain CDR3 of SEQ ID NO: 34.

In the present invention, the binding molecule may be a binding molecule selected from the group consisting of the following (1) to (4):

In the present invention, the binding molecule may be a binding molecule comprising:

a heavy chain variable region consisting of any one amino acid sequence selected from the group consisting of: 11, 19, 27 and 35; and

a light chain variable region consisting of any one of the amino acid sequences selected from the group consisting of: 12, 20, 28 and 36 SEQ ID NOs.

In the present invention, the binding molecule may be a binding molecule selected from the group consisting of:

a binding molecule comprising the heavy chain variable region of SEQ ID NO 11 and the light chain variable region of SEQ ID NO 12;

a binding molecule comprising the heavy chain variable region of SEQ ID NO 19 and the light chain variable region of SEQ ID NO 20;

a binding molecule comprising the heavy chain variable region of SEQ ID NO. 27 and the light chain variable region of SEQ ID NO. 28; and

a binding molecule comprising the heavy chain variable region of SEQ ID No. 35 and the light chain variable region of SEQ ID No. 36.

In the present invention, the binding molecule may further comprise a fragment crystallizable (Fc) region or a constant region. Here, the Fc region may be, or may be derived from, an Fc region of an IgG1, IgG2, IgG3, or IgG4 antibody. Alternatively, the Fc region may be a mixed Fc region (hybrid Fc region).

In the present invention, the Fc region may be that of an IgG1, IgG2, IgG3 or IgG4 antibody of mammalian origin, and preferably of an IgG1, IgG2, IgG3 or IgG4 antibody of human origin. However, the Fc region is not limited thereto.

As an example of the present invention, the Fc region may be, but is not limited to, the mouse-derived IgG2a Fc region shown in SEQ ID NO: 37.

As an example of the present invention, the Fc region may be a mouse-derived immunoglobulin kappa constant region shown in SEQ ID NO: 38.

As an example of the present invention, the Fc region may be the human-derived IgG1 Fc region shown in SEQ ID NO: 39.

As an example of the present invention, the Fc region may be, but is not limited to, the human-derived IgG2 Fc region shown in SEQ ID NO. 40.

As an example of the present invention, the Fc region may be, but is not limited to, the human-derived IgG3 Fc region shown in SEQ ID NO: 41.

As an example of the present invention, the Fc region may be, but is not limited to, the human-derived IgG4 Fc region shown in SEQ ID NO. 42.

As an example of the present invention, the Fc region may be, but is not limited to, the human-derived immunoglobulin kappa constant region shown in SEQ ID NO: 43.

As an example of the present invention, the Fc region may be an immunoglobulin lambda constant region of human origin, but is not limited thereto.

In the present invention, the "hybrid Fc" may be derived from a combination of human IgG subclasses, or a combination of human IgD and IgG. In the case where the mixed Fc is bound to a biologically active molecule, polypeptide or the like, the mixed Fc has not only an effect of increasing the serum half-life of the biologically active molecule but also an effect of increasing the expression level of the polypeptide at the time of expression of the nucleotide sequence encoding the Fc-polypeptide fusion protein.

As an example of the present invention, the mixed Fc region can be represented by SEQ ID NO 44, but is not limited thereto.

In the binding molecules of the invention, the Fc or constant region may be linked to the variable region by a linker. Here, the linker may be linked to the C-terminus of the Fc, and the N-terminus of the binding molecule of the present invention may be linked to the linker. However, the present invention is not limited thereto.

In the present invention, the "linker" may comprise a sequence that can be cleaved by an enzyme overexpressed in a tissue or cell having a disease of interest. If the linker can be cleaved by the above-mentioned overexpressed enzyme, the activity of the polypeptide can be effectively prevented from being reduced due to the Fc portion. In the present invention, examples of the linker may preferably be a peptide linker consisting of 33 amino acids located in parts 282 to 314 of human albumin having the highest content in blood, and more preferably a peptide linker consisting of 13 amino acids located in parts 292 to 304 of human albumin. These portions are the portions that are mostly exposed to the outside in the three-dimensional structure, and thus the possibility of eliciting an immune response in vivo is minimized. However, the joint is not limited thereto.

The binding molecules of the invention may further comprise a heavy chain constant region consisting of an amino acid sequence selected from the group consisting of: SEQ ID NOs 37, 39, 41, 42, 43 and 44.

The binding molecules of the invention may also comprise a light chain constant region consisting of the amino acid sequence shown in SEQ ID NO 38 or 40.

The binding molecules of the invention may further comprise:

a heavy chain constant region consisting of the amino acid sequence shown in SEQ ID NO. 37; and

a light chain constant region consisting of the amino acid sequence shown in SEQ ID NO. 38.

The binding molecules of the invention may further comprise:

a heavy chain constant region consisting of the amino acid sequence shown in SEQ ID NO 39, 41, 42 or 43; and

a light chain constant region consisting of the amino acid sequence shown in SEQ ID NO. 40.

The binding molecules of the invention may further comprise:

a heavy chain constant region consisting of the amino acid sequence shown in SEQ ID NO. 44.

The binding molecules of the invention are selected from the group consisting of:

a binding molecule comprising a heavy chain as set forth in SEQ ID NO 45 and a light chain as set forth in SEQ ID NO 46;

a binding molecule comprising a heavy chain as set forth in SEQ ID NO 47 and a light chain as set forth in SEQ ID NO 48;

a binding molecule comprising a heavy chain as set forth in SEQ ID NO. 49 and a light chain as set forth in SEQ ID NO. 50; and

a binding molecule comprising a heavy chain as shown in SEQ ID NO 51 and a light chain as shown in SEQ ID NO 52.

The binding molecules of the present invention are characterized as antibodies, but are not limited thereto. The antibodies include all monoclonal antibodies, full length antibodies, or antibody fragments that are part of an antibody, have the ability to bind to Lrig-1 protein, and compete with the binding molecules of the invention for binding to an epitope on Lrig-1.

The term "antibody" as used herein refers to protein molecules that serve as receptors specifically recognizing antigens, including immunoglobulin molecules that immunoreact with a particular antigen. For the purposes of the present invention, an antigen may be an Lrig-1 protein present on the surface of regulatory T cells. The antibody preferably can specifically recognize a leucine-rich region or an immunoglobulin-like domain of an Lrig-1 protein, but is not limited thereto.

In the present invention, an "immunoglobulin" has a heavy chain and a light chain, and both the heavy chain and the light chain comprise a constant region and a variable region. The variable regions of both heavy and light chains contain 3 hypervariable regions called complementarity determining regions (hereinafter referred to as "CDRs") and 4 framework regions. CDRs are primarily used to bind epitopes on antigens. The CDRs of each chain are commonly referred to as CDR1, CDR2, and CDR3 in that order from the N-terminus, and are distinguished by the chain in which the particular CDR is located.

Furthermore, the term "monoclonal antibody" as used herein refers to an antibody molecule of single molecular composition that is isolated from a substantially identical population of antibodies and exhibits a single binding specificity and affinity for a particular epitope.

In the present invention, a "full-length antibody" has a structure comprising two full-length light chains and two full-length heavy chains, wherein each light chain is linked to a heavy chain by a disulfide bond, and the full-length antibody includes IgA, IgD, IgE, IgM, and IgG. IgG includes IgG1, IgG2, IgG3 and IgG4 subtypes.

Furthermore, the term "antigen fragment" as used herein refers to a fragment that retains the work of antigen bindingPotential fragments include Fab, Fab ', F (ab')2, Fv, etc. The Fab has a structure comprising the variable regions of the light and heavy chains, the constant region of the light chain and the first constant region of the heavy chain (CH1 domain), and has an antigen binding site. Furthermore, Fab 'differs from Fab in that Fab' has a hinge region comprising at least one cysteine residue at the C-terminus of the heavy chain CH1 domain. Cysteine residues in the hinge region of Fab 'form disulfide bonds, thereby producing F (ab')₂An antibody. Fv (variable fragment) refers to the smallest antibody fragment having only the variable regions of the heavy and light chains. A two-chain fv (dsfv) configured such that the heavy chain variable region and the light chain variable region are linked to each other by a disulfide bond, and a single-chain fv (scfv) configured such that the heavy chain variable region and the light chain variable region are covalently linked to each other, typically by a peptide linker. In the case of using a proteolytic enzyme (e.g., papain or pepsin), the resulting antibody fragment may be Fab or F (ab')₂Fragments, and can be produced by gene recombination techniques.

Furthermore, in the present invention, the antibody may be, but is not limited to, a chimeric antibody, a humanized antibody, a bivalent/bispecific molecule, a small antibody, a domain antibody, a bispecific antibody, an antibody mimetic, a diabody, a triabody, or a tetrabody, or a fragment thereof.

In the present invention, a "chimeric antibody" is an antibody obtained by recombining a variable region of a mouse antibody and a constant region of a human antibody, and the immune response is greatly improved as compared with a mouse antibody.

Furthermore, the term "humanized antibody" as used herein refers to an antibody obtained by modifying the protein sequence of an antibody derived from a non-human species such that the protein sequence resembles a naturally occurring variant of the antibody in humans. For example, a humanized antibody can be prepared as follows. The mouse-derived CDR may be recombined with a human-antibody-derived FR to prepare a humanized variable region, and the humanized variable region may be recombined with a constant region of a preferred human antibody to prepare a humanized antibody. In the present invention, the binding molecule provided may be a bispecific antibody or a bispecific antigen-binding fragment, which is capable of binding to both an Lrig-1 protein and also another protein.

In the present invention, bispecific antibodies and bispecific antigen-binding fragments may comprise a binding molecule of the present invention. As examples of the invention, bispecific antibodies and bispecific antigen-binding fragments comprise an antigen-binding domain capable of binding to an Lrig-1 protein, wherein the antigen-binding domain capable of binding to an Lrig-1 protein may comprise or consist of a binding molecule of the invention.

The bispecific antibodies and bispecific antigen-binding fragments provided by the present invention comprise: an antigen binding domain which is a binding molecule capable of binding to the Lrig-1 protein of the invention; and an antigen binding domain capable of binding another target protein. Here, the antigen binding domain capable of binding another target protein may be an antigen binding domain capable of binding a protein other than the Lrig-1 protein (such as, but not limited to, PD-1 or a cell surface receptor). However, the antigen binding domain is not limited thereto.

Bispecific antibodies and bispecific antigen-binding fragments of the invention may take any suitable form, for example as described in Kontermann MAbs 2012,4(2): 182-. For example, the bispecific antibody or bispecific antigen-binding fragment can be a bispecific antibody conjugate (e.g., IgG2, F (ab ')2, or CovX body), a bispecific IgG or IgG-like molecule (e.g., IgG, scFv4-Ig, IgG-scFv, scFv-IgG, DVD-Ig, IgG-sVD, sVD-IgG2 in 1-IgG, mAb2, or diabatic consensus LC (Tademab consensus LC), an asymmetric bispecific IgG or IgG-like molecule (e.g., kih IgG consensus (LC kih IgG consensus LC), CrossMab, kih IgG-scFab, mAb-Fv, charge pair, or SEED body), a small bispecific antibody molecule (e.g., diabody (Db), dsDb, DART, scDb, tandAb, tandem scFv, tandem dAb/VHH, trisomy, triple head antibody (triple head), Fab-scFv, or F (2') scFv, Bispecific Fc and CH3 fusion proteins (e.g., taFv-Fc, bi-diabody, scDb-CH3, scFv-Fc-scFv, HCAb-VHH, scFv-kih-Fc, or scFv-kih-CH3), or bispecific fusion proteins (e.g., scFv 2-albumin, scDb-albumin, taFv-toxin, DNL-Fab3, DNL-Fab4-IgG, DNL-Fab 4-IgG-cytokine 2). See in particular FIG. 2 of Kontermann MAbs 2012,4(2): 182-19. Bispecific antibodies and bispecific antigen-binding fragments of the invention can be designed and prepared by those skilled in the art.

Methods for producing Bispecific Antibodies in the present invention include the formation of reducing disulfide bonds or non-reducing thioether bonds, as well as chemical crosslinking of Antibodies or antibody fragments, e.g., as described in Segal and Bast,2001, "Production of Bispecific Antibodies", Current Protocols in immunology, 14: IV:2.13:2.13.1-2.13.16, which is incorporated herein by reference in its entirety. For example, a Fab fragment can be chemically cross-linked by, for example, SH-groups of the hinge region using N-succinimidyl-3- (-2-pyridyldithio) -propionate (SPDP) to generate a disulfide-linked bispecific F (ab)2 heterodimer.

In addition, another method of producing bispecific antibodies in the present invention involves the fusion of antibody-producing hybridomas with, for example, polyethylene glycol, to produce tetravalent hybridoma cells capable of secreting bispecific antibodies, e.g., D.M. and Bast, B.J.2001, "production of bispecific antibodies", described in the latest immunological protocols, 14: IV:2.13: 2.13.1-2.13.16.

Bispecific antibodies and bispecific antigen-binding fragments of the invention can also be produced recombinantly, e.g., by expression from a nucleic acid construct encoding an antigen-binding molecule polypeptide, e.g., as described in "antibody engineering: methods and Protocols, second edition (U.S. HumanaPress,2012), Chapter 40: "production of bispecific antibodies: antibodies and Tandem scFv "(Production of bispecific Antibodies: Diabodies and tandm scFv) (Hornig and Farber-Schwarz), or French," How to make bispecific Antibodies "(How to make bispecific Antibodies), MethodsMol.Med.2000; 40:333, 339, both of which are incorporated herein by reference in their entirety.

For example, a DNA construct may be prepared by molecular cloning techniques, said DNA construct comprising: sequences encoding the light and heavy chain variable domains of both antigen binding domains (i.e., the light and heavy chain variable domains capable of binding to the antigen binding domain of PD-1, and the light and heavy chain variable domains capable of binding to the antigen binding domain of another target protein), and sequences encoding suitable linkers or dimerization domains between the antigen binding domains. Subsequently, the recombinant bispecific antibody can be produced by expressing the construct (e.g., in vitro) in a suitable host cell (e.g., a mammalian host cell), and the expressed recombinant bispecific antibody can then optionally be purified.

Antibodies can be produced by affinity maturation methods in which the affinity of the modified antibody for the antigen is improved as compared to the unmodified parent antibody. Affinity matured antibodies can be generated by methods known in the art, e.g., Marks et al, Rio/Technology 10:779-783 (1992); barbas et al, Proc Nat. Acad. Sci. USA 91: 3809-; schier et al, Gene 169:147-155 (1995); yelton et al, J.Immunol.155:1994-2004 (1995); jackson et al, J.Immunol.154(7):3310-159 (1995); and Hawkins et al, J.mol.biol.226:889-896 (1992).

Furthermore, the binding molecules provided by the invention may comprise variants of the amino acid sequences, as long as the variants can specifically bind to the Lrig-1 protein. For example, the amino acid sequence of an antibody may be modified in order to improve the binding affinity and/or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and/or substitutions of amino acid sequence residues of the antibody.

Such amino acid changes can be made based on the relative similarity of the amino acid side-chain substituents, e.g., hydrophobicity, hydrophilicity, charge, and size. Based on analysis of the size, shape and type of the amino acid side-chain substituents, it can be seen that arginine, lysine and histidine are all positively charged residues; alanine, glycine and serine are similar in size; phenylalanine, tryptophan and tyrosine are similar in shape. Thus, based on these considerations, arginine, lysine, and histidine; alanine, glycine and serine; phenylalanine, tryptophan and tyrosine can be considered as biologically functional equivalents.

When introducing the change, the hydropathic index (hydropathic index) of the amino acid may be considered. Each amino acid is assigned a hydropathic index according to its hydrophobicity and charge: isoleucine (+ 4.5); valine (+ 4.2); leucine (+ 3.8); phenylalanine (+ 2.8); cysteine/cystine (+ 2.5); methionine (+ 1.9); alanine (+ 1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The hydropathic-hydrophobic amino acid index is very important in conferring interactive biological functions to proteins. It is known that substitution with amino acids having similar hydropathic indices can maintain similar biological activities in proteins. In the case where a change is introduced with reference to the hydropathic index, substitution is made between amino acids exhibiting a difference in the hydropathic index preferably within. + -.2, more preferably within. + -.1, even more preferably within. + -. 0.5.

It is also well known that substitution between amino acids having similar hydrophilicity values results in proteins of equivalent biological activity. As described in U.S. patent No. 4,554,101, each amino acid residue was given the following hydrophilicity values: arginine (+ 3.0); lysine (+ 3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+ 0.3); asparagine (+ 0.2); glutamine (+ 0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). In the case where a variation is introduced with reference to the hydrophilicity value, substitution may be made between amino acids exhibiting a difference in hydrophilicity value preferably within. + -.2, more preferably within. + -.1, even more preferably within. + -. 0.5.

It is known in The art that amino acid exchanges in Proteins do not completely alter The activity of The molecule (h. neurath, r.l. hill, Proteins, academic press, new york (1979)). The most commonly occurring exchanges are exchanges between the following amino acid residues: Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Gln/Glu.

In view of the above-mentioned variations having biologically equivalent activity, it is to be construed that the binding molecules of the present invention also include sequences having substantial identity to the sequences listed in the sequence listing.

The term "substantial identity" as used herein refers to sequences that match each other to the greatest extent when the sequences of the invention are aligned with any other sequence and that exhibit at least 61% homology, more preferably 70% homology, even more preferably 80% homology, most preferably 90% homology when the aligned sequences are analyzed using algorithms commonly used in the art. Alignment methods for comparing sequences are known in the art. Various methods and algorithms for alignment are disclosed in Smith and Waterman, adv.appl.math.2:482 (1981); needleman and Wunsch, J.mol.Bio.48:443 (1970); pearson and Lipman, Methods in mol. biol.24:307-31 (1988); higgins and Sharp, Gene 73:237-44 (1988); higgins and Sharp, CABIOS 5:151-3 (1989); corpet et al, Nuc. acids Res.16:10881-90 (1988); huang et al, Comp.appl.BioSci.8:155-65 (1992); and Pearson et al, meth.mol.biol.24:307-31 (1994). The basic search tool for NCBI local sequence alignment (BLAST) is available from the National Bioinformatics Center (NBCI) and the like (Altschul et al, J.mol.biol.215:403-10(1990)), which can be used in conjunction with sequencing programs on the Internet, such as blastp, blastm, blastx, tblastn, and tblastx. BLSAT is accessible from http:// www.ncbi.nlm.nih.gov/BLAST/ACCESS. The sequence homology comparison method using this program (http:// www.ncbi.nlm.nih.gov/BLAST/BLAST _ help. html) can be identified online.

In the present invention, the binding molecule, preferably an antibody, can be produced by conventional methods for producing antibodies, but also by affinity maturation.

The term "affinity maturation" as used herein refers to the process of producing antibodies with increased affinity for an antigen from activated B cells during an immune response. For the purposes of the present invention, affinity maturation allows the production of antibodies or antibody fragments based on the principles of mutation and selection in the same process as occurs in nature.

The binding molecules, preferably antibodies, provided by the invention can activate the function of immune cells, in particular regulatory T immune cells (Treg cells); increasing the number of Treg cells; and (3) regulating immune tolerance, thereby effectively preventing, improving or treating immune-related diseases.

In the present invention, the "immune-related disease" may be a disease induced by over-activation and expression of various immune cells and inflammatory cells. Immune-related diseases may include, for example, autoimmune diseases; and graft versus host disease; organ transplant rejection; asthma; atopic diseases; or acute or chronic inflammatory diseases, but is not limited thereto.

Further, in the present invention, the "autoimmune disease" may be, but is not limited to, one or more selected from the group consisting of: rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behcet's disease, Sjogren's syndrome, Guillain-Barre syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibrositis (fibrositis), and polyarteritis nodosa.

According to another embodiment of the invention, there is provided a nucleic acid molecule encoding a binding molecule provided by the invention.

As known to the person skilled in the art, the nucleic acid molecules of the invention include all nucleic acid molecules obtained by converting the amino acid sequence of the binding molecules provided by the invention into a polynucleotide sequence. Thus, various polynucleotide sequences can be prepared by Open Reading Frames (ORFs), and all such polynucleotide sequences are also included in the nucleic acid molecules of the present invention.

According to yet another embodiment of the present invention, there is provided an expression vector into which the isolated nucleic acid molecule provided by the present invention is inserted.

In the present invention, a "vector" is a nucleic acid molecule capable of transporting another nucleic acid linked thereto. One type of vector is a "plasmid," which refers to a circular double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a phage vector. Yet another type of vector is a viral vector in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., a bacterial vector having a bacterial origin of replication is an episomal mammalian vector). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors". Expression vectors which can be used in recombinant DNA techniques are usually in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably, as plasmids are the most commonly used form of vector.

In the present invention, specific examples of the expression vector may be selected from, but are not limited to, the following groups: pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, Ti vectors, which are widely used commercially; sticking particles; phages, such as lambda phage, M13 phage, Mu phage, P1 phage, P22, phage Q μ phage, T-doublet phage, T2 phage, T3 phage, T7 phage; a plant virus. As an expression vector, any expression vector known to those skilled in the art can be used in the present invention, and the expression vector is selected according to the properties of the target host cell. The vector may be introduced into the host cell by calcium phosphate transfection, viral infection, DEAE-dextran mediated transfection, lipofection, or electroporation. However, the present invention is not limited thereto, and a person skilled in the art can adopt and use introduction methods suitable for the expression vector and host cell used. The vector may preferably comprise at least one selectable marker. However, the present invention is not limited thereto, and selection may be performed using a vector not containing a selection marker depending on whether or not the product is produced. The selection of the selectable marker depends on the target host cell, which can be accomplished using methods known to those skilled in the art, and thus the present invention is not limited in this regard.

To facilitate purification of the nucleic acid molecules of the invention, the tag sequence may be inserted into and fused to an expression vector. Tags include, but are not limited to, a hexahistidine tag, a hemagglutinin tag, a myc tag, or a flag tag, and any tag known to those skilled in the art to facilitate purification can be used in the present invention.

In yet another embodiment of the invention, host cell lines transfected with the expression vectors provided herein are provided.

In the present invention, "host cell" includes a single cell or cell culture, which can be or has been one or more incorporation of polypeptide insert carrier recipients. Host cells include progeny of a single host cell, and the progeny may not necessarily be identical (morphologically or complementary in genomic DNA) to the original parent cell due to natural, accidental, or deliberate mutation. Host cells include cells transfected in vivo with one or more polynucleotides herein.

In the present invention, the host cell may include a cell of mammalian, plant, insect, fungal or cellular origin, and may be, for example, a bacterial cell such as Escherichia coli (E.coli), Streptomyces (Streptomyces), Salmonella typhimurium (Salmonella typhimurium); fungal cells, such as yeast cells and pichia pastoris (Pichiapastoris); insect cells such as Drosophila (Drosophila) and Spodoptera (Spodoptera) Sf9 cells; animal cells, such as Chinese Hamster Ovary (CHO) cells, SP2/0 (mouse myeloma), human lymphoblastoid cells, COS, NSO (mouse myeloma), 293T, Bos melanoma cells, HT-1080, Baby Hamster Kidney (BHK) cells, Human Embryonic Kidney (HEK) cells, or PERC.6 (human retinal cells); or a plant cell. However, the host cell is not limited thereto, and any cell known to those skilled in the art to be usable as a host cell line may be used.

Yet another embodiment of the present invention provides an antibody-drug conjugate (ADC) comprising the antibody provided by the present invention and a drug.

In the present invention, the antibody-drug conjugate refers to a form in which a drug is bound to the N-terminal amino acid residue of the heavy chain and/or light chain of an antibody, particularly a form in which a drug is bound to the N-terminal α -amine group of the heavy chain and/or light chain of an antibody.

The drug may be in the form of a reactive group capable of reacting and cross-linking with α -amine group, and also includes a form of a reactive group capable of reacting and cross-linking with α -amine group and linker attachment.

In the present invention, examples of the reactive group capable of reacting with α -amine group and crosslinking are not particularly limited in type as long as the reactive group can react with α -amine group at the N-terminal of heavy chain or light chain of the antibody and crosslink.

In the present invention, the drug includes any type of drug as long as the drug can treat a disease targeted by an Lrig-1 antibody, and preferably may be a therapeutic agent for an immune-related disease such as an autoimmune disease, graft-versus-host disease, organ transplant rejection, asthma, an atopic disease, an acute or chronic inflammatory disease, or the like.

Yet another embodiment of the present invention provides a pharmaceutical composition for preventing or treating immune-related diseases, comprising the binding molecule or antibody-drug conjugate (ADC) provided in the present invention as an active ingredient.

In the present invention, the "immune-related disease" may be a disease induced by over-activation and expression of various immune cells and inflammatory cells. Immune-related diseases may include, for example, autoimmune diseases; graft versus host disease; organ transplant rejection; asthma; atopic diseases or acute or chronic inflammatory diseases, but are not limited thereto.

Further, in the present invention, the "autoimmune disease" may be, but is not limited to, one or more selected from the group consisting of: rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, crohn's disease, beset's disease, sjogren's syndrome, guillain-barre syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibrositis, and polyarteritis nodosa.

Meanwhile, in the present invention, "prevention" may include, but is not limited to, any action of blocking symptoms of a disease, or inhibiting or delaying symptoms using the pharmaceutical composition of the present invention.

Further, in the present invention, "treatment" may include, but is not limited to, any act of alleviating or ameliorating symptoms of a disease using the pharmaceutical composition of the present invention.

In the present invention, the pharmaceutical composition may be characterized in the form of a capsule, tablet, granule, injection, ointment, powder, or granule, and the pharmaceutical composition may be characterized as being targeted to a human.

In the present invention, the pharmaceutical composition may be separately prepared in the form of oral preparations such as powders, granules, capsules, tablets and aqueous suspensions, external preparations, suppositories and sterile injectable solutions according to conventional methods and used. However, the pharmaceutical composition is not limited thereto. The pharmaceutical compositions of the present invention may also comprise a pharmaceutically acceptable carrier. As pharmaceutically acceptable carriers, binders, glidants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavoring agents, and the like may be used for oral administration; buffers, preservatives, analgesics, solubilizers, isotonizing agents, stabilizers and the like may be mixed for injection; bases, excipients, lubricants, preservatives and the like may be used for topical administration. The formulation of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with the above-mentioned pharmaceutically acceptable carrier. For example, for oral administration, the pharmaceutical compositions may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. For injection, the pharmaceutical compositions may be formulated in unit-dose ampoules or in multi-dose forms. Alternatively, the pharmaceutical composition may be formulated into a solution, suspension, tablet, capsule, sustained-release formulation, or the like.

Meanwhile, as examples of carriers, diluents or excipients suitable for preparing formulations, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like can be used. In addition, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like may also be included.

Routes of administration of the pharmaceutical compositions of the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal routes. Oral or parenteral administration is preferred.

In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intravesicular (intraburst), intrasternal, intradural, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions of the present invention may also be administered rectally in the form of suppositories.

The pharmaceutical composition of the present invention may vary depending on a variety of factors, including the activity of a certain compound used, the age, body weight, general health, sex, diet, administration time, administration route, excretion rate, drug combination, and the severity of certain diseases to be prevented or treated. The dosage of the pharmaceutical composition may vary depending on the condition, body weight, severity of disease, pharmaceutical form, administration route and duration of the patient, and may be appropriately selected by those skilled in the art. The pharmaceutical composition may be administered in an amount of 0.0001 to 50mg/kg or 0.001 to 50mg/kg per day. Administration may be once a day or several times a day. This dosage is not intended to limit the scope of the invention in any way. The pharmaceutical composition of the present invention may be formulated in the form of pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries or suspensions.

The invention has the advantages of

Specific binding molecules, preferably antibodies, of the Lrig-1 protein of the invention can activate the function of immune cells, particularly regulatory T immune cells (Treg cells); increasing the number of Treg cells; and (2) modulating immune tolerance, thereby effectively preventing, ameliorating or treating immune-related diseases induced by over-activation and expression of various immune cells and inflammatory cells, such as autoimmune diseases, graft-versus-host diseases, organ transplant rejection, asthma, atopic diseases, or acute or chronic inflammatory diseases.

Furthermore, the specific binding molecules, preferably antibodies, of the Lrig-1 protein of the invention have the following advantages: the binding molecules are able to target the Lrig-1 protein more efficiently and with very good binding capacity than previous commercially available anti-Lrig-1 antibodies.

Brief Description of Drawings

Fig. 1 shows the structure of Lrig-1 protein according to an embodiment of the present invention.

Fig. 2 shows the structure of Lrig-1 protein according to an embodiment of the present invention.

Fig. 3 shows the prediction results of epitopes of Lrig-1 protein according to an embodiment of the present invention.

Fig. 4 shows the prediction results of epitopes of Lrig-1 protein according to an embodiment of the present invention.

FIG. 5 shows the expression level of Lrig-1mRNA according to one embodiment of the present invention.

FIG. 6 shows the expression level of Lrig-1mRNA according to an embodiment of the present invention.

FIG. 7 shows the expression level of Lrig-1mRNA according to an embodiment of the present invention.

FIG. 8 shows the expression levels of Lrig-1, Lrig-2 and Lrig-3mRNA according to one embodiment of the present invention.

FIG. 9 shows the results obtained by comparing the expression levels of Lrig-1 protein in regulatory T cells and non-regulatory T cells according to an embodiment of the present invention.

FIG. 10 shows the expression of Lrig-1 protein on the surface of regulatory T cells according to one embodiment of the present invention.

Fig. 11 shows the results obtained by analyzing the binding ability of monoclonal antibodies specific to Lrig-1 protein (a7, C8, E7, and G3) to Lrig-1 protein according to an embodiment of the present invention.

Fig. 12 shows the results obtained by analyzing the mechanism of Lrig-1 protein-specific monoclonal antibodies (a7, C8, E7, and G3) to modulate the phosphorylation of the Lrig-1 protein-induced Stat3 in regulatory T cells, according to an embodiment of the present invention.

Fig. 13 shows experimental design of therapeutic effect of monoclonal antibodies specific to Lrig-1 protein (a7, C8, E7, and G3) on autoimmune diseases according to an embodiment of the present invention.

Fig. 14 shows the results obtained by analyzing the therapeutic effect of the monoclonal antibodies specific to Lrig-1 protein (a7, C8, E7, and G3) on autoimmune diseases according to an embodiment of the present invention.

Detailed Description

One embodiment of the present invention provides a binding molecule selected from the following (1) to (4):

(1) a binding molecule comprising a heavy chain variable region comprising heavy chain CDR1 of SEQ ID No. 5, CDR2 of SEQ ID No. 6 and CDR3 of SEQ ID No. 7; the light chain variable region comprises the light chain CDR1 of SEQ ID NO. 8, the light chain CDR2 of SEQ ID NO. 9 and the light chain CDR3 of SEQ ID NO. 10;

(2) a binding molecule comprising a heavy chain variable region comprising heavy chain CDR1 of SEQ ID No. 13, CDR2 of SEQ ID No. 14 and CDR3 of SEQ ID No. 15 and a light chain variable region; the light chain variable region comprises the light chain CDR1 of SEQ ID NO. 16, the light chain CDR2 of SEQ ID NO. 17 and the light chain CDR3 of SEQ ID NO. 18;

(3) a binding molecule comprising a heavy chain variable region comprising heavy chain CDR1 of SEQ ID No. 21, heavy chain CDR2 of SEQ ID No. 22 and heavy chain CDR3 of SEQ ID No. 23 and a light chain variable region; the light chain variable region comprises the light chain CDR1 of SEQ ID NO. 24, the light chain CDR2 of SEQ ID NO. 25 and the light chain CDR3 of SEQ ID NO. 26;

(4) a binding molecule comprising a heavy chain variable region comprising heavy chain CDR1 of SEQ ID No. 29, heavy chain CDR2 of SEQ ID No. 30 and heavy chain CDR3 of SEQ ID No. 31 and a light chain variable region; the light chain variable region comprises the light chain CDR1 of SEQ ID NO. 32, the light chain CDR2 of SEQ ID NO. 33 and the light chain CDR3 of SEQ ID NO. 34.

The present invention will be described in more detail below by way of examples. These examples are only for describing the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

Examples

Preparation example 1 cell culture of T cell subsets

To identify whether Lrig-1 protein is expressed only in regulatory T cells (tregs), subsets of T cells Th0, Th1, Th2, Th17 and iTreg were prepared. Unlike naturally isolated ntregs, iTreg refers to cells that are artificially induced to differentiate in a medium containing the following composition.

Naive T cells obtained from mouse spleen were first isolated such that RPMI1640(Invitrogen Gibco, Greend island, N.Y.) nutrient medium containing 10% fetal bovine serum (FBS; Hai clone-HyClone, Roots, Utah) further contained the individual components of Table 1 below and was incubated at 37 deg.C, 5% CO₂The incubator of (a) was cultured for 72 hours, thereby inducing the differentiation of a subpopulation of T cells into corresponding cells.

[ Table 1]

[ example 1]Structural analysis of Lrig-1

The three-dimensional structure of the extracellular domain of the Lrig-1 protein is predicted to produce antibodies specific for the Lrig-1 protein, a surface protein of regulatory T cells.

First, in order to predict the base sequence of an epitope, the three-dimensional spatial structure of the extracellular domain (ECD) of Lrig-1 protein was predicted using tools of Uniprot (http:// www.uniprot.org) and RCSB protein database (http:// www.rcsb.org/pdb), thereby determining the structure of ECD. Then, the results are shown in fig. 1 and 2.

As shown in fig. 1, a total of 15 leucine-rich regions, LRR1 to LRR15, are present in the Lrig-LRR domain (amino acid sequence at positions 41 to 494) of the extracellular domain of the Lrig-1 protein. Each LRR domain consists of 23 to 27 amino acids, of which 3 to 5 leucines are present.

Furthermore, as shown in FIG. 2, three immunoglobulin-like domains are present in the amino acid sequence of positions 494 to 781 of the Lrig-1 protein in the extracellular domain of the Lrig-1 protein.

[ example 2]Prediction of amino acid sequence of Lrig-1 epitope

The prediction of the above base sequence was performed using an Ellipro server (http:// tools. iedb. org/Ellipro /), which is epitope prediction software based on the structure of the Lrig-1 protein. The Ellipro search engine is utilized because it corresponds to the most reliable search engine known in the existing predictive epitope algorithms.

The extracellular domain analyzed in example 1 was imported into epitope prediction software, and the continuous or discontinuous amino acid sequences of the predicted epitopes are shown in FIGS. 3 and 4.

As shown in fig. 3 and 4, a total of 22 contiguous epitope amino acid sequences were predicted, and a total of 8 non-contiguous epitope amino acid sequences were predicted.

Production examples 1 to 4]Production of monoclonal antibodies to Lrig-1 protein

Antibodies specific for the Lrig-1 protein of the invention are generated. The antibodies of the invention are not produced by specific epitopes, but rather as antibodies capable of binding to any site on the Lrig-1 protein.

To produce antibodies, cells expressing the Lrig-1 protein are generated. More specifically, a DNA fragment corresponding to SEQ ID NO 2 and pcDNA (hygro) were cleaved with a cleaving enzyme, incubated at 37 ℃ and ligated to produce pcDNA into which the DNA sequence of Lrig-1 protein was inserted. The pcDNA thus generated, inserted into SEQ ID NO:2, was introduced into L cells by transfection, so that the Lrig-1 protein could be expressed on the surface of the L cells.

Light and heavy chain amino acid sequences capable of binding to cell surface-expressed Lrig-1 were selected from a human scFv library, thereby selecting a total of eight heavy and light chains.

Selected heavy and light chain amino acid sequences were fused to mlgG2a Fc region to produce monoclonal antibodies. The sequences of the monoclonal antibodies are shown in table 2 below.

[ Table 2]

[ example 3]Identification of specific expression of Lrig-1mRNA in regulatory T cells

It was verified whether the Lrig-1 protein could be used as a biomarker for regulatory T cells.

For validation, CD4 was isolated from mouse spleen by CD4 beads using a magnetic activated cell sorting Method (MACS)⁺T cells. Subsequently, regulatory T (CD 4) was isolated using a Fluorescence Activated Cell Sorter (FACS) using CD25 antibody⁺CD25⁺T) cells and non-regulatory T (CD 4)⁺CD25^-T) cells. For each cell and the cells differentiated in preparation example 1, mRNA was extracted using Trizol and genomic RNA was extracted using gDNA extraction kit (qiagen) according to the protocol provided by the manufacturerRemoving gDNA. The gDNA-depleted mRNA was synthesized into cDNA using the BDsprint cDNA Synthesis kit (cloning technologies).

Real-time polymerase chain reaction (RT PCR) was performed to quantitatively identify the expression level of Lrig-1mRNA in the cDNA.

Real-time polymerase chain reaction was performed using primers shown in table 3 below using SYBR Green (molecular probes) according to the protocol provided by the manufacturer, including 95 ℃, 3 minutes, 61 ℃, 15 seconds, 72 ℃, 30 seconds, 40 cycles, relative gene expression levels were calculated using Δ Δ CT method, and normalized using HPRT. The results are shown in FIGS. 5 to 8.

[ Table 3]

As can be seen in FIG. 5, Lrig-1 is at regulatory T (CD 4)⁺CD25⁺Expression ratio in T) cells to non-regulatory T (CD 4)⁺CD25^-T) cells were 18.1 times higher. This is about 10-fold higher than the expression levels of the previously known regulatory T cell markers lang 3 and Ikzf 4. Furthermore, as shown in fig. 6 and 7, Lrig-1mRNA expression was significantly higher in regulatory T cells compared to other types of immune cells, and in particular, in naturally isolated regulatory T cells (ntregs) compared to induced regulatory T cells (iTreg cells).

Furthermore, as shown in FIG. 8, the expression of Lrig-1 was highest among Lrig-1, Lrig-2 and Lrig-3 corresponding to the Lrig family.

From the above results, it can be seen that the Lrig-1 protein of the present invention is specifically expressed in regulatory T cells, particularly naturally occurring regulatory T cells.

[ example 4 ]]Identification of specific expression of Lrig-1 protein in regulatory T cells

It was determined whether the Lrig-1 protein expressed from Lrig-1mRNA was specifically expressed only in regulatory T cells.

FOXP 3-RFP-knock-in mice were used, FOXP3-RFP obtained by coupling the Red Fluorescent Protein (RFP) to the FOXP3 promoter (transcription factor specific for regulatory T cells), and CD4 was isolated from mouse spleen using CD4 beads using the magnetically activated cell sorting Method (MACS)⁺T cells. Subsequently, the RFP protein was used for separation by Fluorescence Activated Cell Sorter (FACS), thereby obtaining regulatory T (CD 4)⁺RFP⁺T) cells and non-regulatory T (CD 4)⁺RFP^-T) cells. The expression level of Lrig-1 was measured using a fluorescence activated cell sorter by staining each cell with purchased Lrig-1 antibody and staining the negative control with isotype matched control antibody. The results are shown in FIG. 9.

As shown in FIG. 9, the non-regulatory T cells indicated by the dotted line showed almost the same expression level of Lrig-1 as the negative control, whereas a large number of cells having a high expression level of Lrig-1 were present in the regulatory T cells.

As can be seen from the above results, the Lrig-1 protein of the present invention is specifically expressed in regulatory T cells.

[ example 5]Identification of specific expression of Lrig-1 protein on the surface of regulatory T cells

From the viewpoint that, in order to be a target of cell therapy, Lrig-1 protein must be expressed on the surface of regulatory T cells so that target therapy can be performed more efficiently, it was determined whether Lrig-1 protein is expressed on the surface of regulatory T cells.

Each of the differentiated T cell subsets of preparation example 1 was stained with anti-CD 4-APC and anti-Lrig-1-PE antibodies, and the Lrig-1 expression level on the surface of each cell was detected using a Fluorescence Activated Cell Sorter (FACS). The results are shown in FIG. 10.

As shown in fig. 10, the expression amount of Lrig-1 in activated T cells, Th1 cells, Th2 cells, Th17 cells and naive T cells was 0.77 to 15.3, whereas the expression amount of Lrig-1 in differentiation-induced T cells (iTreg cells) was as high as 83.9.

As can be seen from the above results, the Lrig-1 protein of the present invention is not only specifically expressed in regulatory t (Treg) cells, but is also expressed at a higher level, particularly on the surface of Treg cells.

[ example 6]Evaluation of binding Capacity of the antibody of the present invention to Lrig-1 protein

To identify whether the monoclonal antibodies of the present invention produced in production examples 1 to 8 recognized Lrig-1 well, each of the antibodies in production examples 1 to 8 was bound to L cells stably expressing Lrig-1. Then, a secondary antibody which is coupled to the erfour 670 and is capable of recognizing a mouse antibody was added thereto, and then the binding ability of the monoclonal antibody to the Lrig-1 protein was analyzed using FACS. The results are shown in FIG. 11.

As shown in fig. 11, it was found that all of the monoclonal antibodies specific to Lrig-1 protein (a7, C8, E7 and G3) of the present invention efficiently recognized and bound to Lrig-1 protein present on the surface of L cells.

[ example 7]Modulation of signal transduction pathways in Treg cells by antibodies of the invention

To analyze how the monoclonal antibodies of the present invention produced in production examples 1 to 8 affect the signal transduction pathway in Treg cells through Lrig-1 protein, Lrig-1 present on the surface of Treg cells was stimulated by treating Treg cells with the antibodies of production examples 1 to 8, and then the tyrosine phosphorylation levels of Stat3 protein present in the stimulated Treg cells were analyzed by phosphotyrosine immunoblotting. The results are shown in FIG. 12.

As shown in fig. 12, it was found that the monoclonal antibodies specific to Lrig-1 protein of the present invention (a7, C8, E7 and G3) increased the phosphorylation of Stat3 to the same level as that of Th17 cells.

[ example 8]Therapeutic effects of the antibodies of the invention on autoimmune diseases

To identify the therapeutic effect of the monoclonal antibodies of the invention (A7, C8, E7 and G3) produced in production examples 1 to 4 on autoimmune diseases, RAG-1-^/Adoptive transfer of CD45RB (high) cells in mice, thereby inducing Inflammatory Bowel Disease (IBD), an autoimmune disease. Then, the antibodies of production examples 1 to 4 were injected intraperitoneally at 200. mu.g/mouse, and then the therapeutic effects on autoimmune diseases were analyzed. The results are shown in FIG. 13.

As shown in fig. 13, it was found that the Lrig-1 protein-specific monoclonal antibodies (a7, C8, E7 and G3) of the present invention significantly inhibited the inflammatory bowel disease-induced weight loss effect in mice.

It can be seen that the monoclonal antibody specific to the Lrig-1 protein of the present invention is effective in preventing, ameliorating or treating immune-related diseases such as autoimmune diseases, graft-versus-host diseases, organ transplant rejection, asthma, atopic diseases, or acute or chronic inflammatory diseases caused by the over-activation and expression of various immune cells and inflammatory cells.

Although the present invention has been described in detail above, the scope of the present invention is not limited thereto. It will be apparent to those skilled in the art that various modifications and changes may be made without departing from the technical idea of the present invention described in the claims.

INDUSTRIAL APPLICABILITY

The present invention relates to binding molecules capable of specifically binding to leucine-rich and immunoglobulin-like domain 1(Lrig-1) proteins present on the surface of regulatory T cells (Treg cells), in particular for the prevention or treatment of immune-related diseases, such as autoimmune diseases, graft-versus-host diseases, organ transplant rejection, asthma, atopic diseases or acute or chronic inflammatory diseases, and uses thereof.

Sequence listing

<110> Goodt T cells Co., Ltd

Specific binding molecule of <120> LRIG-1 protein and application thereof

<130>DPB172433.k01

<150>KR 10-2017-0049854

<151>2017-04-18

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Ser Trp Thr Arg Ser Leu Asn Leu Ser Tyr Asn Lys Leu Ser Glu Ile

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Asp Pro Ala Gly Phe Glu Asp Leu Pro Asn Leu Gln Glu Val Tyr Leu

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

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His Val Val Ser Leu Phe Leu Gln His Asn Lys Ile Arg Ser Val Glu

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Gly Ser Gln Leu Lys Ala Tyr Leu Ser Leu Glu Val Leu Asp Leu Ser

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Leu Asn Asn Ile Thr Glu Val Arg Asn Thr Cys Phe Pro His Gly Pro

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Pro Ile Lys Glu Leu Asn Leu Ala Gly Asn Arg Ile Gly Thr Leu Glu

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

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Ser Lys Asn Arg Ile Thr Gln Leu Pro Val Arg Ala Phe Lys Leu Pro

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Arg Leu Thr Gln Leu Asp Leu Asn Arg Asn Arg Ile Arg Leu Ile Glu

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Gly Leu Thr Phe Gln Gly Leu Asn Ser Leu Glu Val Leu Lys Leu Gln

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

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Asn Asn Ser Ile Ala Arg Ile His Arg Lys Gly Trp Ser Phe Cys Gln

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Glu Glu Ser Leu Ala Glu Leu Ser Ser Leu Ser Val Leu Arg Leu Ser

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His Asn Ser Ile Ser His Ile Ala Glu Gly Ala Phe Lys Gly Leu Arg

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Glu Ile Asp Ser Ala Ala Phe Glu Asp Leu Thr Asn Leu Gln Glu Val

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Tyr Leu Asn Ser Asn Glu Leu Thr Ala Ile Pro Ser Leu Gly Ala Ala

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Ser Ile Gly Val Val Ser Leu Phe Leu Gln His Asn Lys Ile Leu Ser

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

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Leu Ser Ser Asn Asn Ile Thr Glu Ile Arg Ser Ser Cys Phe Pro Asn

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

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Arg Leu Ser Lys Asn Arg Ile Thr Gln Leu Pro Val Lys Ala Phe Lys

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Leu Pro Arg Leu Thr Gln Leu Asp Leu Asn Arg Asn Arg Ile Arg Leu

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Ile Glu Gly Leu Thr Phe Gln Gly Leu Asp Ser Leu Glu Val Leu Arg

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Val Asn Ser Gly Ser Leu Tyr Gly Leu Thr Ala Leu His Gln Leu His

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Leu Ser Asn Asn Ser Ile Ser Arg Ile Gln Arg Asp Gly Trp Ser Phe

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Cys Gln Lys Leu His Glu Leu Ile Leu Ser Phe Asn Asn Leu Thr Arg

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Leu Asp Glu Glu Ser Leu Ala Glu Leu Ser Ser Leu Ser Ile Leu Arg

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Leu Ser His Asn Ala Ile Ser His Ile Ala Glu Gly Ala Phe Lys Gly

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Leu Lys Ser Leu Arg Val Leu Asp Leu Asp His Asn Glu Ile Ser Gly

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Thr Ile Glu Asp Thr Ser Gly Ala Phe Thr Gly Leu Asp Asn Leu Ser

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

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Phe Ser Gly Leu Glu Ser Leu Glu His Leu Asn Leu Gly Glu Asn Ala

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Ile Arg Ser Val Gln Phe Asp Ala Phe Ala Lys Met Lys Asn Leu Lys

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Glu Leu Tyr Ile Ser Ser Glu Ser Phe Leu Cys Asp Cys Gln Leu Lys

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Trp Leu Pro Pro Trp Leu Met Gly Arg Met Leu Gln Ala Phe Val Thr

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Ala Thr Cys Ala His Pro Glu Ser Leu Lys Gly Gln Ser Ile Phe Ser

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Val Leu Pro Asp Ser Phe Val Cys Asp Asp Phe Pro Lys Pro Gln Ile

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Ile Thr Gln Pro Glu Thr Thr Met Ala Val Val Gly Lys Asp Ile Arg

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Phe Thr Cys Ser Ala Ala Ser Ser Ser Ser Ser Pro Met Thr Phe Ala

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Trp Lys Lys Asp Asn Glu Val Leu Ala Asn Ala Asp Met Glu Asn Phe

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Ala His Val Arg Ala Gln Asp Gly Glu Val Met Glu Tyr Thr Thr Ile

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

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Thr Val Asn Val Leu Pro Ser Phe Thr Lys Ile Pro His Asp Ile Ala

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Ile Arg Thr Gly Thr Thr Ala Arg Leu Glu Cys Ala Ala Thr Gly His

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Pro Asn Pro Gln Ile Ala Trp Gln Lys Asp Gly Gly Thr Asp Phe Pro

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Ala Ala Arg Glu Arg Arg Met His Val Met Pro Asp Asp Asp Val Phe

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Phe Ile Thr Asp Val Lys Ile Asp Asp Met Gly Val Tyr Ser Cys Thr

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Ala Gln Asn Ser Ala Gly Ser Val Ser Ala Asn Ala Thr Leu Thr Val

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Leu Glu Thr Pro Ser Leu Ala Val Pro Leu Glu Asp Arg Val Val Thr

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Val Gly Glu Thr Val Ala Phe Gln Cys Lys Ala Thr Gly Ser Pro Thr

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Pro Arg Ile Thr Trp Leu Lys Gly Gly Arg Pro Leu Ser Leu Thr Glu

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Arg His His Phe Thr Pro Gly Asn Gln Leu Leu Val Val Gln Asn Val

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Met Ile Asp Asp Ala Gly Arg Tyr Thr Cys Glu Met Ser Asn Pro Leu

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gccttctggg ggctgtctaa gatgcacgtg ctgcacctgg agtacaacag tctggtggaa 720

gtgaacagtg gctccctcta tggcctcaca gccctgcacc agctgcacct cagcaacaac 780

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ctgtccttca acaacctcac gcggctggat gaggagagtc tagcggagtt gagcagcctc 900

agtatcctgc gcctcagtca caacgccatc agtcacattg ctgaaggcgc cttcaaggga 960

ctcaagagtc tgcgggtctt ggacctggac cataacgaga tctcgggtac aatcgaggat 1020

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ggagagaatg caatcaggtc tgtccagttt gatgcctttg caaagatgaa gaaccttaaa 1200

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ctgaagggcc agagcatttt ctcagtgctg ccagacagct ttgtgtgtga tgactttcca 1380

aagccacaga tcatcaccca gcctgagacg accatggctg tggtgggcaa ggacatccgt 1440

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gaagtgatgg agtataccac tatcctgcac ctccgtcacg tcacctttgg gcacgagggc 1620

cgctaccagt gtatcatcac aaaccacttt ggctccacat actcccacaa agccaggctc 1680

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accacagccc gcctcgagtg tgctgccacg ggccacccta accctcagat tgcctggcag 1800

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Gly Ser Trp Asp Tyr Ser Leu Ser Ala

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Gly Ser Thr Trp Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val

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Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

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Phe Ser Gly Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly

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Leu Glu Trp Val Ser Leu Ile Tyr Pro Asp Ser Gly Asn Lys Tyr Tyr

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

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

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Val Tyr Tyr Cys Ala Arg Asp Ala Gly Leu Ser Trp Ala Gly Ala Phe

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Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

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Gly Ser Thr Trp Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly

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Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn

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Ile Gly Ser Asn Tyr Val Thr Trp Tyr Gln Gln Leu Pro Gly Thr Ala

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Pro Lys Leu Leu Ile Tyr Ser Asp Ser His Arg Pro Ser Gly Val Pro

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

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Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp

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

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Val Leu

130

<210>13

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Asn Tyr Tyr Met Ser

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<213> Artificial sequence (Artificial sequence)

<400>14

Gly Ile Ser Pro Gly Asp Ser Ser Thr

1 5

<210>15

<211>13

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>15

Lys Gly Leu Tyr Ser Asn Pro Asn Glu Pro Phe Asp Tyr

1 5 10

<210>16

<211>13

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>16

Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Ser

1 5 10

<210>17

<211>4

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>17

Asp Asp Ser Gln

<210>18

<211>9

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>18

Gly Thr Trp Asp Tyr Ser Leu Asn Gly

1 5

<210>19

<211>141

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>19

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Trp Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val

20 2530

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

35 40 45

Phe Ser Asn Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ser Gly Ile Ser Pro Gly Asp Ser Ser Thr Tyr Tyr

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

130 135 140

<210>20

<211>130

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>20

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Trp Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly

20 25 30

Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn

35 40 45

Ile Gly Ser Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala

50 55 60

Pro Lys Leu Leu Ile Tyr Asp Asp Ser Gln Arg Pro Ser Gly Val Pro

65 70 75 80

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

85 90 95

Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp

100 105 110

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

115 120 125

Val Leu

130

<210>21

<211>5

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>21

Ser Tyr Asp Met Ser

1 5

<210>22

<211>9

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>22

Gly Ile Ser Pro Asp Gly Ser Asn Ile

1 5

<210>23

<211>19

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>23

Lys Val Gly Leu Arg Cys Arg Tyr Glu Ala Cys Ser Tyr Ala Tyr Gly

1 5 10 15

Met Asp Val

<210>24

<211>13

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>24

Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Ser

1 5 10

<210>25

<211>4

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>25

Ser Asp Ser His

<210>26

<211>9

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>26

Ala Thr Trp Asp Ser Ser Leu Asn Gly

1 5

<210>27

<211>136

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>27

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Trp Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val

20 25 30

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

35 40 45

Phe Ser Ser Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ser Gly Ile Ser Pro Asp Gly Ser Asn Ile Tyr Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Val Tyr Tyr Cys Ala Lys Val Gly Leu Arg Cys Arg Tyr Glu Ala Cys

115 120 125

Ser Tyr Ala Tyr Gly Met Asp Val

130 135

<210>28

<211>130

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>28

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Trp Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly

20 25 30

Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn

35 40 45

Ile Gly Ser Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala

50 55 60

Pro Lys Leu Leu Ile Tyr Ser Asp Ser His Arg Pro Ser Gly Val Pro

65 70 75 80

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

85 90 95

Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp

100 105 110

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

115 120 125

Val Leu

130

<210>29

<211>5

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>29

Asn Tyr Asp Met Ser

1 5

<210>30

<211>9

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>30

Ser Ile Ser Pro Ser Ser Gly Ser Ile

1 5

<210>31

<211>13

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>31

Lys Asp Leu Asp Ala Phe Trp Arg Pro Ser Phe Asp Tyr

1 5 10

<210>32

<211>13

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>32

Thr Gly Ser Ser Ser Asn Ile Gly Asn Asn Asn Val Asn

1 510

<210>33

<211>4

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>33

Ser Asp Ser His

<210>34

<211>9

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>34

Gly Ser Trp Asp Asp Ser Leu Ser Ala

1 5

<210>35

<211>141

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>35

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Trp Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val

20 25 30

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

35 40 45

Phe Ser Asn Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ser Ser Ile Ser Pro Ser Ser Gly Ser Ile Tyr Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Val Tyr Tyr Cys Ala Lys Asp Leu Asp Ala Phe Trp Arg Pro Ser Phe

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

130 135 140

<210>36

<211>130

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>36

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Trp Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly

20 25 30

Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn

35 40 45

Ile Gly Asn Asn Asn Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala

50 55 60

Pro Lys Leu Leu Ile Tyr Ser Asp Ser His Arg Pro Ser Gly Val Pro

65 70 75 80

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

85 90 95

Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp

100 105 110

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

115 120 125

Val Leu

130

<210>37

<211>328

<212>PRT

<213> mouse (Mus musculus)

<400>37

Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr

1 5 10 15

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

20 25 30

Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val

35 40 45

His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser

5055 60

Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys

65 70 75 80

Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu

85 90 95

Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala

100 105 110

Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile

115 120 125

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

130 135 140

Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val

145 150 155 160

Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp

165 170 175

Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln

180 185 190

Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp

195 200 205

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

210 215220

Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr

225 230 235 240

Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu

245 250 255

Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr

260 265 270

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

275 280 285

Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr

290 295 300

Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys

305 310 315 320

Ser Phe Ser Arg Thr Pro Gly Lys

325

<210>38

<211>107

<212>PRT

<213> mouse (Mus musculus)

<400>38

Arg Thr Val Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

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

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210>39

<211>330

<212>PRT

<213> Intelligent (Homo sapiens)

<400>39

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

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>40

<211>107

<212>PRT

<213> Intelligent (Homo sapiens)

<400>40

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser GlyThr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

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

65 70 75 80

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

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210>41

<211>326

<212>PRT

<213> Intelligent (Homo sapiens)

<400>41

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210>42

<211>377

<212>PRT

<213> Intelligent (Homo sapiens)

<400>42

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro

100 105 110

Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg

115 120 125

Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys

130 135 140

Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

145 150 155 160

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

165 170 175

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

180 185 190

Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr

195 200 205

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

210 215 220

Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu

325 330 335

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

340 345 350

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln

355 360 365

Lys Ser Leu Ser Leu Ser Pro Gly Lys

370 375

<210>43

<211>327

<212>PRT

<213> Intelligent (Homo sapiens)

<400>43

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 9095

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

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

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

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

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

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

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

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

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

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

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

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

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>44

<211>245

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>44

Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys

1 5 10 15

Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His

20 25 30

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

35 40 45

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

50 5560

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Leu Ser Leu Gly Lys

245

<210>45

<211>469

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>45

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Trp Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val

20 25 30

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

35 40 45

Phe Ser Gly Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ser Leu Ile Tyr Pro Asp Ser Gly Asn Lys Tyr Tyr

65 70 75 80

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

85 90 95

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

100105 110

Val Tyr Tyr Cys Ala Arg Asp Ala Gly Leu Ser Trp Ala Gly Ala Phe

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Ala

130 135 140

Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser

145 150 155 160

Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val

165 170 175

Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe

180 185 190

Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr

195 200 205

Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala

210 215 220

His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly

225 230 235 240

Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu

245 250 255

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

260265 270

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

275 280 285

Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val

290 295 300

Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser

305 310 315 320

Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met

325 330 335

Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala

340 345 350

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

355 360 365

Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln

370 375 380

Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr

385 390 395 400

Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr

405 410 415

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

420 425430

Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser

435 440 445

Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser

450 455 460

Arg Thr Pro Gly Lys

465

<210>46

<211>237

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>46

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Trp Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly

20 25 30

Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn

35 40 45

Ile Gly Ser Asn Tyr Val Thr Trp Tyr Gln Gln Leu Pro Gly Thr Ala

50 55 60

Pro Lys Leu Leu Ile Tyr Ser Asp Ser His Arg Pro Ser Gly Val Pro

65 70 75 80

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

85 9095

Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp

100 105 110

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

115 120 125

Val Leu Arg Thr Val Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser

130 135 140

Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn

145 150 155 160

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

165 170 175

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

180 185 190

Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu

195 200 205

Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser

210 215 220

Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

225 230 235

<210>47

<211>469

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>47

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Trp Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val

20 25 30

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

35 40 45

Phe Ser Asn Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ser Gly Ile Ser Pro Gly Asp Ser Ser Thr Tyr Tyr

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Ala

130 135 140

Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser

145 150 155 160

Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val

165 170 175

Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe

180 185 190

Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr

195 200 205

Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala

210 215 220

His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly

225 230 235 240

Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu

245 250 255

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

260 265 270

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

275 280 285

Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val

290 295 300

Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser

305 310 315 320

Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met

325 330 335

Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala

340 345 350

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

355 360 365

Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln

370 375 380

Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr

385 390 395 400

Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr

405 410 415

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

420 425 430

Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser

435 440 445

Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser

450 455 460

Arg Thr Pro Gly Lys

465

<210>48

<211>237

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>48

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Trp Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly

20 25 30

Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn

35 40 45

Ile Gly Ser Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala

50 55 60

Pro Lys Leu Leu Ile Tyr Asp Asp Ser Gln Arg Pro Ser Gly Val Pro

65 70 75 80

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

85 90 95

Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp

100 105 110

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

115 120 125

Val Leu Arg Thr Val Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser

130 135 140

Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn

145 150 155 160

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

165 170 175

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

180 185 190

Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu

195 200 205

Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser

210 215 220

Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

225 230 235

<210>49

<211>475

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>49

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Trp Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val

20 25 30

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

35 40 45

Phe Ser Ser Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ser Gly Ile Ser Pro Asp Gly Ser Asn Ile Tyr Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Val Tyr Tyr Cys Ala Lys Val Gly Leu Arg Cys Arg Tyr Glu Ala Cys

115 120 125

Ser Tyr Ala Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr

130 135 140

Val Ser Ser Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys

145 150 155 160

Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly

165 170 175

Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser

180 185 190

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr

195 200 205

Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser

210 215 220

Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys

225 230 235 240

Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys

245 250 255

Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro

260 265 270

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

275 280 285

Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser

290 295 300

Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His

305 310 315 320

Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile

325 330 335

Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn

340 345 350

Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys

355 360 365

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

370 375 380

Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe

385 390 395 400

Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu

405 410 415

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

420 425 430

Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg

435 440 445

Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His

450 455 460

Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

465 470 475

<210>50

<211>237

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>50

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Trp Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly

20 25 30

Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn

35 40 45

Ile Gly Ser Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala

50 55 60

Pro Lys Leu Leu Ile Tyr Ser Asp Ser His Arg Pro Ser Gly Val Pro

65 70 75 80

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

85 90 95

Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp

100 105 110

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

115 120 125

Val Leu Arg Thr Val Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser

130 135 140

Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn

145 150 155 160

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

165 170 175

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

180 185 190

Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu

195 200 205

Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser

210 215 220

Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

225 230 235

<210>51

<211>469

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>51

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Trp Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val

20 25 30

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

35 40 45

Phe Ser Asn Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ser Ser Ile Ser Pro Ser Ser Gly Ser Ile Tyr Tyr

65 70 7580

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

85 90 95

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

100 105 110

Val Tyr Tyr Cys Ala Lys Asp Leu Asp Ala Phe Trp Arg Pro Ser Phe

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Ala

130 135 140

Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser

145 150 155 160

Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val

165 170 175

Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe

180 185 190

Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr

195 200 205

Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala

210 215 220

His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly

225 230 235240

Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu

245 250 255

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

260 265 270

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

275 280 285

Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val

290 295 300

Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser

305 310 315 320

Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met

325 330 335

Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala

340 345 350

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

355 360 365

Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln

370 375 380

Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr

385 390 395400

Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr

405 410 415

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

420 425 430

Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser

435 440 445

Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser

450 455 460

Arg Thr Pro Gly Lys

465

<210>52

<211>237

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>52

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Trp Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly

20 25 30

Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn

35 40 45

Ile Gly Asn Asn Asn Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala

50 55 60

Pro Lys Leu Leu Ile Tyr Ser Asp Ser His Arg Pro Ser Gly Val Pro

65 70 75 80

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

85 90 95

Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp

100 105 110

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

115 120 125

Val Leu Arg Thr Val Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser

130 135 140

Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn

145 150 155 160

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

165 170 175

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

180 185 190

Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu

195 200 205

Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser

210 215 220

Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

225 230 235

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Specific binding molecules for LRIG-1 proteins and uses thereof

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