KR102114191B1 - Antibody specifically binding to the N-terminal region of lysyl-tRNA synthetase exposed at cell outer membrane - Google Patents

Abstract

The present invention relates to an antibody that specifically binds to the N-terminal region of a lysyl-tRNA synthetase exposed to the outer cell membrane and the use thereof, and more specifically, a specific CDR (complementarity determining region) sequence described herein. An antibody or fragment thereof having a lysyl-tRNA synthetase (KRS, Lysyl-tRNA synthetase) N-terminal specifically binding to an epitope comprising the sequence of SEQ ID NO: 97, and cancer metastasis of the antibody or fragment It is for suppression purpose and cancer diagnosis use.
Since the antibody or fragments according to the present invention have a very good specific binding ability to the KRS N-terminal region exposed to the outer membrane, a disease known as a disease (eg, cancer) accompanying the specific behavior of KRS Available for diagnosis. In addition, since it is specifically targeted to the KRS N-terminal region in vivo, it can be used as a therapeutic agent because it has an excellent effect of inhibiting cancer metastasis by inhibiting the interaction between the laminin receptor and the KRS N-terminal region, so it can be used as a therapeutic agent, making it industrially applicable Big.

Description

Antibody specifically binding to the N-terminal region of lysyl-tRNA synthetase exposed at cell outer membrane}

The present invention relates to an antibody that specifically binds to the N-terminal region of a lysyl-tRNA synthetase exposed to the outer cell membrane and the use thereof, and more specifically, a specific CDR (complementarity determining region) sequence described herein. An antibody or fragment thereof having a lysyl-tRNA synthetase (KRS, Lysyl-tRNA synthetase) N-terminal specifically binding to an epitope comprising the sequence of SEQ ID NO: 97, and cancer metastasis of the antibody or fragment For suppression and cancer diagnosis

Through recent studies, human KRS (lysyl-tRNA synthetase), which is generally present in the cytosol, is translocated to the plasma membrane (cell membrane) and 67LR (67-kDa laminin receptor) present in the plasma membrane It has been demonstrated that it promotes the migration of tumor (or cancer) cells by interacting with) and affects metastasis of cancer (Dae Gyu Kim et al., Chemical inhibition of prometastatic lysyl-tRNA synthetase-laminin receptor interaction) , Nat Chem Biol. 2014 Jan; 10 (1): 29-34, Dae Gye Kim et.al.Interaction of two translational components, lysyl-tRNA synthetase and p40 / 37LRP, in plasma membrane promotes laminin-dependent cell migration, FASEB J. (2012) 26, 4142-4159). Human KRS (Genebank Accession No. NP_005539.1, etc.) has an N-terminal extension (1-72), an anticodon-binding domain (73-209) and a catalytic domain (220-220). 597). Human KRS is an enzyme essential for protein synthesis and is usually present in the cytoplasm in a multi-tRNA synthetase complex (MSC). However, after laminin signaling, p38 MAPK phosphorylates KRS at the T52 residue, and KRS migrates to the cell membrane to protect 67LR from ubiquitin-mediated degradation. It has also been reported that KRS migrated to the cell membrane promotes cancer metastasis by stabilizing and interacting with 67LR associated with cancer metastasis.

At this time, the fact that Myc-KRS41-597 (ΔN), in which 40 terminal residues of the N-terminal extension (N-ext) is deleted, is not localized to the plasma membrane, the KRS N-ext region moves to the cell membrane ( translocation). It has also been found that in relation to cancer metastasis, the KRS N-ext region is specifically involved in their binding in the interaction with 67LR. In order to use this fact for therapeutic or diagnostic purposes, it is necessary to specifically target a specific target position (especially KRS N-ext) in a protein according to the characteristics of various domain regions constituting the KRS protein.

However, despite its importance as a biomarker for ARS (aminoacyl tRNA synthetase) including KRS, ARSs have many similarities in protein structure, so antibodies obtained by an immune response from animals show cross-reactivity to other ARSs, and high-sensitivity antibodies are generated. Often it doesn't.

Accordingly, the present inventors were studying to produce an antibody that specifically binds to the KRS N-terminal region exposed to the outer cell membrane, while antibodies having a specific CDR (complementarity determining region) sequence described herein are KRS N-terminal region In addition to showing a very high binding specificity (specificity) and binding affinity (affinity), the present invention was completed by confirming that it inhibits cancer metastasis in vivo .

Accordingly, an object of the present invention is to provide an antibody or fragment thereof that specifically binds to an epitope comprising the sequence of SEQ ID NO: 97 from the N-terminal of Lysyl-tRNA synthetase (KRS). will be.

Another object of the present invention is to provide a polynucleotide encoding the antibody or fragment thereof according to the present invention, a recombinant expression vector containing the polynucleotide, and a cell transformed with the recombinant expression vector.

Another object of the present invention, (a) transforming a host cell with the recombinant expression vector; (b) culturing the transformed host cell to produce an antibody or fragment thereof; And (c) obtaining an antibody produced from a host cell or a fragment thereof, an antibody specifically binding to the N-terminus of Lysyl-tRNA synthetase (KRS) exposed to the outer cell membrane. Or to provide a method for producing the fragment.

Another object of the present invention is to provide a pharmaceutical composition for inhibiting cancer metastasis comprising the antibody or fragment thereof according to the present invention as an active ingredient.

Another object of the present invention is to provide a composition for diagnosing cancer comprising the antibody according to the present invention or a fragment thereof as an active ingredient.

In order to achieve the above object, the present invention is an antibody that specifically binds to an epitope comprising the sequence of SEQ ID NO: 97 among the N-terminals of Lysyl-tRNA synthetase (KRS) or Provide that fragment.

In order to achieve another object of the present invention, the present invention provides a polynucleotide encoding the antibody or fragment thereof according to the present invention, a recombinant expression vector containing the polynucleotide, and cells transformed with the recombinant expression vector. .

In order to achieve another object of the present invention, the present invention (a) transforming a host cell with the recombinant expression vector; (b) culturing the transformed host cell to produce an antibody or fragment thereof; And (c) obtaining an antibody produced from a host cell or a fragment thereof, an antibody specifically binding to the N-terminus of Lysyl-tRNA synthetase (KRS) exposed to the outer cell membrane. Or it provides a method for producing the fragment.

In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for inhibiting cancer metastasis comprising the antibody or fragment thereof as an active ingredient.

In order to achieve another object of the present invention, the present invention provides a composition for diagnosing cancer comprising the antibody or a fragment thereof as an active ingredient.

Hereinafter, the present invention will be described in detail.

In the present invention, "lysyl-tRNA synthetase (KRS) N-terminal region exposed to the outer cell membrane" is an extracellular region when the KRS generated in the cell moves and is located in the cell membrane (or plasma membrane). It refers to a specific sequence exposed on the cell membrane surface, and may mean a part or a full-length sequence of the 1 to 72 amino acid region of the KRS N-terminal. In addition, the KRS N-terminal region has a sequence similarity between species, and may be characterized in particular comprising an amino acid sequence represented by SEQ ID NO: 97. Preferably, the sequence includes the sequence represented by SEQ ID NO: 75 in humans, the sequence represented by SEQ ID NO: 113 in the mouse, and the sequence represented by SEQ ID NO: 114 in the rat.

In the present invention, "KRS" refers to any KRS fragment sequence comprising a full-length polypeptide or N-terminal extention, known as lysyl R & A synthetase. Since the antibody or fragments according to the present invention specifically detect the KRS N-terminal region exposed to the outer cell membrane as described above, any KRS fragment sequence comprising the aforementioned KRS full-length polypeptide or N-terminal region also Can be detected. The specific sequence of the KRS is not particularly limited as long as it includes the polypeptide represented by SEQ ID NO: 75 and is known in the art as a lysyl R & A synthetase. For example, the KRS of the present invention is derived from human ( homo sapiens ). As NCBI (Genbank) Accession No. NP_005539.1 and the like, and derived from a mouse ( Mus musculus ) NCBI (Genbank) Accession No. NP_444322.1, etc., and is derived from rat ( Rattus norvegicus ), NCBI (Genbank) Accession No. XP_006255692.1, and the like, and may refer to the following sequence information in addition to, but is not limited to: XP_005004655.1 (guinea-pig: Cavia porcellus ), XP_021503253.1 (gerbil, Meriones unguiculatus ), XP_002711778.1 (rabbit, Oryctolagus cuniculus ), XP_536777.2 (dog, Canis lupus familiaris ), XP_003126904.2 (swine, *? * Sus scrofa ), XP_011755768.1 (monkey, Macaca nemestrina ), XP_008984479.1 (marmoset, Callithrix jacchus ), XP_019834275.1 (cow, Bos indicus ), XP_511115.2 (chimpanzee, Pan troglodytes ). Most preferably, it may be a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 76 (Genbank Accession No. NP_005539.1).

In the present invention, 'antibody' is also called immunoglobulin (Ig), and is a generic term for proteins involved in bio-immunity by selectively acting on antigens. Whole antibodies found in nature usually consist of two pairs of light chains (LC) and heavy chains (HCs), which are polypeptides of multiple domains, or two pairs of these HC / LCs The basic structure is the basic unit. There are five types of heavy chains constituting mammalian antibodies, which are represented by the Greek letters α, δ, ε, γ, and μ, and different types of antibodies, such as IgA, IgD, IgE, IgG, and IgM, respectively, depending on the type of heavy chain. Will constitute. There are two types of light chains constituting mammalian antibodies, represented by λ and κ.

The heavy and light chains of an antibody are structurally divided into variable regions and constant regions according to the variability of amino acid sequences. The constant region of the heavy chain is composed of 3 or 4 heavy chain constant regions such as CH1, CH2 and CH3 (IgA, IgD and IgG antibodies) and CH4 (IgE and IgM antibodies) depending on the type of antibody, and the light chain has one constant region It consists of CL. The variable region of the heavy chain and the light chain consists of one domain of the heavy chain variable region (VH) or light chain variable region (VL), respectively. The light and heavy chains of each variable region and the constant region are aligned side by side and linked by one covalent disulfide bond, and the heavy chains of the two molecules bound by the light chain are linked through two covalent disulfide bonds to form the entire antibody. To form. The whole antibody specifically binds to the antigen through the variable regions of the heavy and light chains, and since the whole antibody is composed of two pairs of heavy and light chains (HC / LC), the whole antibody of one molecule has two variable regions. This results in a bivalent monospecific binding to the same two antigens.

The variable region containing the antibody-binding site is subdivided into a framework region (FR) with less sequence variability and a complementary determining region (CDR), a hypervariable region with high sequence variability. do. In the VH and VL, three CDRs and four FRs are arranged in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in the direction from N-terminal to C-terminal, respectively. It is a site in which the CDR having the highest sequence variability in the variable region of the antibody directly binds to the antigen, and is the most important for the antigen specificity of the antibody.

The present invention is lysyl- tRNA  Synthetase ( KRS , Lysyl - tRNA synthetase ) Provides an antibody or fragment thereof that specifically binds an epitope comprising the sequence of SEQ ID NO: 97 from the N-terminus.

In the present invention, 'epitope' refers to a protein determinant capable of specifically binding to an antibody. Epitopes usually consist of surface groups of molecules, such as amino acids or sugar side chains, and generally have specific three-dimensional structural properties and specific charge properties. Conformational and non-conformational epitopes are distinguished in that in the presence of a denaturing solvent, binding to the conformational epitope is lost, but not for non-conformational epitopes. Epitopes are amino acid residues that are directly involved in binding (also called immunogenic components of the epitope) and other amino acid residues that are not directly related to binding, such as amino acid residues that are effectively blocked by specific antigen binding peptides (i.e. Specific antigen-binding peptide (which is present in the footprint).

Preferably, a site to which the N3 monoclonal antibody of the present invention derived from the KRS N-terminal sequence binds, and if it is a contiguous region comprising the amino acid represented by SEQ ID NO: 97 (klsknelkrrlka), the specific sequence is not particularly limited. 13 to 52 including the amino acid sequence of SEQ ID NO: 97, more preferably 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 amino acid sequence.

Preferably, the epitope of the present invention may be an amino acid sequence represented by SEQ ID NO: 75, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100 or SEQ ID NO: 101 derived from a human KRS N-terminal, mouse KRS It may be an amino acid sequence represented by SEQ ID NO: 113, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105 and SEQ ID NO: 106 derived from the N-terminal, SEQ ID NO: 114, derived from the rat KRS N-terminal It may be an amino acid sequence represented by SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110 and SEQ ID NO: 111. More preferably, it may be a sequence (SEQ ID NO: 75, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100 and SEQ ID NO: 101) comprising the amino acid sequence of 15 to 29 of the human KRS N-terminal region represented by SEQ ID NO: 75, , Most preferably 15 to 42 amino acid sequence of the human KRS N-terminal region represented by SEQ ID NO: 75 (SEQ ID NO: 101).

The antibody or fragment thereof that specifically binds to the KRS N-terminal region exposed to the outer cell membrane provided by the present invention is

SEQ ID NO: 1, SEQ ID NO: 13, comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 25 and SEQ ID NO: 37 Heavy chain  Complementarity Determination 1 ( CDR1 ); SEQ ID NO: 3, SEQ ID NO: 15, comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 27 and SEQ ID NO: 39 Heavy chain  Complementarity determining part 2 ( CDR2 ); SEQ ID NO: 5, SEQ ID NO: 17, comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 29 and SEQ ID NO: 41 Heavy chain  Heavy chain variable region (VH) comprising complementarity determining region 3 (CDR3) and

SEQ ID NO: 7, comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 31 and SEQ ID NO: 43 Light chain  Complementarity Determination 1 ( CDR1 ); SEQ ID NO: 9, SEQ ID NO: 21, comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 33 and SEQ ID NO: 45 Light chain  Complementarity determining part 2 ( CDR2 ); SEQ ID NO: 11, SEQ ID NO: 23, comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 35 and SEQ ID NO: 47 Light chain  Light chain variable region (VL) comprising complementarity determining region 3 (CDR3); It characterized in that it comprises a.

Antibodies composed of the CDR sequences have excellent ability to specifically bind to the KRS N-terminal region exposed to the outer cell membrane. This is well illustrated in the specification examples of the present invention. In an embodiment of the present invention, in order to construct a scFv fragment that specifically binds to the KRS N-terminal region exposed to the outer cell membrane, initiation of primary screening through scFv phage library screening, indirect ELISA (secondary screening). After 5 experiments of Western blot (3rd screening), immunoprecipitation (4th screening), and immunofluorescence staining (5th screening), scFv fragments showing high binding specificity and binding affinity for KRS N-terminal binding It was selected. At the first screening through scFv phage library screening, a total of 1920 scFv clones were screened, but after the 5 steps of screening, four types of N3 scFv, N5 scFv, N7 scFv, and N9scFv fragments were finally selected. . In addition, by converting the scFv to IgG Antibodies of N3 IgG, N5 IgG, N7 IgG, and N9 IgG were prepared, and it was confirmed that the antibodies also showed high binding specificity in KRS N-terminal binding.

The antibody or fragment thereof that specifically binds to the KRS N-terminal region exposed to the outer cell membrane according to the present invention is preferably an antibody comprising the following CDR constructs of the heavy and light chain variable regions (i), (ii), (iii) and (iv) show the CDR combinations of the N3, N5, N7 and N9 antibodies of the Examples, respectively:

(i) heavy chain complementarity determining region 1 comprising an amino acid sequence represented by SEQ ID NO: 1, heavy chain complementarity determining region 2 comprising an amino acid sequence represented by SEQ ID NO: 3 and heavy chain complementarity comprising an amino acid sequence represented by SEQ ID NO: 5 Light chain complementarity determining region 1 comprising light chain complementarity determining region 2 comprising SEQ ID NO: 3 and amino acid sequence represented by SEQ ID NO: 7, light chain complementarity determining region 2 comprising SEQ ID NO: 9 and SEQ ID NO: 11 A light chain variable region comprising a light chain complementarity determining region 3 comprising an amino acid sequence;

(ii) heavy chain complementarity determining region 1 comprising the amino acid sequence represented by SEQ ID NO: 13, heavy chain complementarity determining region 2 comprising the amino acid sequence represented by SEQ ID NO: 15 and heavy chain complementarity comprising the amino acid sequence represented by SEQ ID NO: 17 Light chain complementarity determining region 1 comprising light chain complementarity determining region 2 comprising SEQ ID NO: 19 and amino acid sequence represented by SEQ ID NO: 19, light chain complementarity determining region 2 comprising SEQ ID NO: 21 and SEQ ID NO: 23 A light chain variable region comprising a light chain complementarity determining region 3 comprising an amino acid sequence;

(iii) heavy chain complementarity determining region 1 comprising an amino acid sequence represented by SEQ ID NO: 25, heavy chain complementarity determining region 2 comprising an amino acid sequence represented by SEQ ID NO: 27 and heavy chain complementarity comprising an amino acid sequence represented by SEQ ID NO: 29 Light chain complementarity determining region 1 comprising light chain complementarity determining region 3 comprising SEQ ID NO: 31 and light chain complementarity determining region 2 comprising amino acid sequence shown by SEQ ID NO: 33 and SEQ ID NO: 35 A light chain variable region comprising a light chain complementarity determining region 3 comprising an amino acid sequence; And

(iv) heavy chain complementarity determining region 1 comprising the amino acid sequence represented by SEQ ID NO: 37, heavy chain complementarity determining region 2 comprising the amino acid sequence represented by SEQ ID NO: 39 and heavy chain complementarity comprising the amino acid sequence represented by SEQ ID NO: 41 Light chain complementarity determining region 1 comprising light chain complementarity determining region 2 comprising SEQ ID NO: 3, amino acid sequence represented by SEQ ID NO: 43, light chain complementarity determining region 2 comprising SEQ ID NO: 45 and SEQ ID NO: 47 A light chain variable region comprising a light chain complementarity determining region 3 comprising an amino acid sequence; Antibodies or fragments comprising a heavy chain variable region and a light chain variable region selected from the group consisting of.

Most preferably, the antibody or fragment thereof according to the present invention is characterized by comprising a heavy chain variable region and a light chain variable region as follows: In the antibody or fragment thereof, the heavy chain variable region is SEQ ID NO: 49 (N3 VH ), SEQ ID NO: 53 (N5 VH), SEQ ID NO: 57 (N7 VH) and amino acid sequence selected from the group consisting of SEQ ID NO: 61 (N9 VH), the light chain variable region SEQ ID NO: 51 (N3 VL), sequence Amino acid sequence selected from the group consisting of SEQ ID NO: 55 (N5 VL), SEQ ID NO: 59 (N7 VL) and SEQ ID NO: 63 (N9 VL).

The antibody comprising the heavy chain variable region (VH) and the light chain variable region (VL) specifically comprises a heavy chain and a sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85 and SEQ ID NO: 89. It may be an antibody characterized by consisting of a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87 and SEQ ID NO: 91.

Most preferably, a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 77 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 79; A heavy chain comprising an amino acid sequence represented by SEQ ID NO: 81 and a light chain comprising an amino acid sequence represented by SEQ ID NO: 83; A heavy chain comprising the amino acid sequence represented by SEQ ID NO: 85 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 87; It is an antibody comprising a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 89 and a light chain comprising an amino acid sequence represented by SEQ ID NO: 91.

The 'antibody that specifically binds to the KRS N-terminal region exposed to the outer cell membrane' according to the present invention is not limited in its kind, as long as it has the above-described CDR combination or VH and VL combination. As a specific example, the antibody may be selected from the group consisting of IgG, IgA, IgM, IgE and IgD, and may be preferably an IgG antibody.

The antibody of the present invention may be a monoclonal antibody or a polyclonal antibody as long as it has the above-described CDR combination or VH and VL combination that specifically binds to the KRS N-terminal region. It is preferred that the heavy chain and light chain of the amino acid sequence is a monoclonal antibody that is a population of antibodies that are substantially identical.

The antibody of the present invention may be derived from any animal, including mammals, birds, etc., including humans, preferably human-derived, or human-derived antibodies and animals of other species. It may be a chimeric antibody comprising a portion of an antibody. That is, the present invention includes all chimeric antibodies, humanized antibodies, and human antibodies, and may preferably be human antibodies.

In addition, in the present invention, the fragment of the antibody refers to a fragment of the antibody that maintains the antigen-specific binding capacity of the entire antibody, preferably the fragment is at least 20%, 50%, 70 of the KRS N-terminal binding affinity of the parent antibody %, 80%, 90%, 95% or 100% or more. Specifically, it may be in the form of Fab, F (ab) 2, Fab ', F (ab') 2, Fv, diabody, scFv, and the like.

Fab (fragment antigen-binding) is an antigen-binding fragment of an antibody, and is composed of one variable domain and a constant domain of heavy and light chains, respectively. F (ab ') 2 is a fragment produced by hydrolysis of an antibody with pepsin, and the two Fabs are linked by disulfide bonds in a heavy chain hinge. F (ab ') is a monomeric antibody fragment in which heavy chain hinges are added to Fabs separated by reducing disulfide bonds of F (ab') 2 fragments. Fv (variable fragment) is an antibody fragment composed of only the variable regions of the heavy and light chains. The scFv (single chain variable fragment) is a recombinant antibody fragment in which the heavy chain variable region (VH) and the light chain variable region (VL) are linked by a flexible peptide linker. Diabody refers to a fragment in which the VH and VL of the scFv are linked by a very short linker and cannot bind to each other, and forms a dimer by combining with the VL and VH of other scFvs of the same type, respectively.

For the purposes of the present invention, the fragment of the antibody is not limited in structure or form as long as it maintains binding specificity to the KRS N-terminal region exposed to the outer cell membrane, but may be preferably scFv. The scFv according to the present invention has a CDR configuration specific to the above-described KRS N-terminal region, or VH and VL, and the sequence is particularly limited if the C-terminal of VH and the N-terminal of VL are linked via a linker. Does not work. If the linker is known in the art as a linker applied to scFv, its type is not particularly limited, but may preferably be a peptide comprising an amino acid sequence represented by SEQ ID NO: 65. Accordingly, the scFv of the present invention specifically comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 67 (N3 scFv ), SEQ ID NO: 69 (N5 scFv ), SEQ ID NO: 71 (N7 scFv ) and SEQ ID NO: 73 (N9 scFv ) . May be

Antibodies or fragments thereof of the invention may contain conservative amino acid substitutions (referred to as conservative variants of antibodies) that do not substantially alter their biological activity.

In addition, the antibody or fragment thereof of the present invention described above may be conjugated with an enzyme, a fluorescent substance, a radioactive substance and a protein, but is not limited thereto. In addition, methods for conjugating such substances to antibodies are well known in the art.

Also seen  The present invention provides a polynucleotide encoding the antibody or fragment thereof according to the present invention described above.

Polynucleotides may be described herein as oligonucleotides or nucleic acids, and are generated using DNA molecules (eg, cDNA or genomic DNA), RNA molecules (eg, mRNA), and nucleotide analogs. Analogs of the DNA or RNA (eg, peptide nucleic acids and non-naturally occurring nucleotide analogs) and hybrids thereof The polynucleotides are single-stranded or double-stranded (double stranded).

The polynucleotide refers to a base sequence encoding an antibody consisting of a heavy chain and a light chain having a CDR configuration specific to the KRS N-terminal region described above, or a configuration of VH and VL. If the polynucleotide of the present invention encodes an antibody or a fragment thereof of the present invention, the sequence is not particularly limited, and the sequence of the polynucleotide encoding the CDR sequence described above in the antibody according to the present invention described above is particularly limited. Although not preferably, SEQ ID NO: 2 (heavy chain CDR1), SEQ ID NO: 4 (heavy chain CDR2), SEQ ID NO: 6 (heavy chain CDR3), SEQ ID NO: 8 (light chain CDR1), SEQ ID NO: 10 (light chain CDR2), SEQ ID NO: 12 (light chain) CDR3), SEQ ID NO: 14 (heavy chain CDR1), SEQ ID NO: 16 (heavy chain CDR2), SEQ ID NO: 18 (heavy chain CDR3), SEQ ID NO: 20 (light chain CDR1), SEQ ID NO: 22 (light chain CDR2), SEQ ID NO: 24 (light chain CDR3) , SEQ ID NO: 26 (heavy chain CDR1), SEQ ID NO: 28 (heavy chain CDR2), SEQ ID NO: 30 (heavy chain CDR3), SEQ ID NO: 32 (light chain CDR1), SEQ ID NO: 34 (light chain CDR2), SEQ ID NO: 36 (light chain CDR3), sequence SEQ ID NO: 38 (heavy chain CDR1), SEQ ID NO: 40 (heavy chain CDR2), SEQ ID NO: 42 (heavy chain CDR3), SEQ ID NO: 44 ( It may be one that includes the nucleotide sequence shown in chain CDR1), SEQ ID NO: 46 (light chain CDR2) or SEQ ID NO: 48 (light chain CDR3).

 In addition, the polynucleotide encoding the above-mentioned VH and VL in the antibody according to the present invention is not particularly limited in sequence, but preferably SEQ ID NO: 50 (VH), SEQ ID NO: 52 (VL), SEQ ID NO: 54 (VH), sequence It may be one comprising a nucleotide sequence represented by SEQ ID NO: 56 (VL), SEQ ID NO: 58 (VH), SEQ ID NO: 60 (VL), SEQ ID NO: 62 (VH) or SEQ ID NO: 64 (VL).

In addition, the polynucleotide encoding the fragment of the antibody preferably includes any one nucleotide sequence selected from the group consisting of SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72 and SEQ ID NO: 74 encoding the scFv according to the present invention. It may be.

The polynucleotide encoding the antibody or fragment thereof of the present invention can be obtained by methods well known in the art. For example, oligonucleotide synthesis techniques well known in the art, for example, polymerase chain reaction (PCR), etc. Can be synthesized.

The present invention provides a recombinant expression vector comprising a polynucleotide encoding an antibody or fragment thereof according to the present invention.

In the present invention, 'recombinant' may be used interchangeably with 'genetic manipulation', and in a state of nature using molecular cloning experiment techniques such as modification, cutting, and linking of genes. It means producing a gene that does not exist.

In the present invention, 'expression' means that a protein or nucleic acid is produced in a cell.

In the present invention, 'recombinant expression vector' is a vector capable of expressing a desired protein or nucleic acid (RNA) in a suitable host cell, and is an essential regulation operably linked to allow expression of a polynucleotide (gene) insert. Refers to a gene construct containing urea. 'Operably linked' is a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein or RNA to perform a general function, and the gene is controlled by the expression control sequence. It means that it is connected so that it can be expressed. The 'expression control sequence' refers to a DNA sequence that controls the expression of a polynucleotide sequence operably linked in a specific host cell. Such regulatory sequences include promoters for conducting transcription, any operator sequence for regulating transcription, sequences encoding suitable mRNA ribosomal binding sites, sequences regulating the termination of transcription and translation, initiation codons, termination codons, polyadenylation Signals and enhancers.

The recombinant expression vector of the present invention is not particularly limited as long as it is a vector commonly used in the cloning field, and examples thereof include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors and viral vectors. The plasmids include E. coli-derived plasmids (pBR322, pBR325, pUC118 and pUC119, pET-22b (+)), Bacillus subtilis-derived plasmids (pUB110 and pTP5), and yeast-derived plasmids (YEp13, YEp24 and YCp50). As the virus, animal viruses such as retrovirus, adenovirus or vaccinia virus, insect viruses such as baculovirus, and the like can be used.

Therefore, in the recombinant expression vector according to the present invention, a polynucleotide encoding an antibody or a fragment thereof composed of heavy and light chains having the above-described CDR or VH and VL capable of specifically binding to the KRS N-terminal region is appropriate. Refers to a gene construct operably linked to be expressed in a host cell.

The polynucleotides encoding the heavy and light chains of the antibody according to the present invention may be included in separate recombinant expression vectors or may be included in one recombinant expression vector.

The present invention provides cells transformed with the aforementioned recombinant expression vectors.

The cell of the present invention is not particularly limited as long as it can be used to express a polynucleotide encoding an antibody or fragment thereof included in the recombinant expression vector of the present invention. Cells (host cells) transformed with the recombinant expression vector according to the present invention include prokaryotes (eg, E. coli), eukaryotes (eg, yeast or other fungi), plant cells (eg, tobacco or tomato). Plant cells), animal cells (eg, human cells, monkey cells, hamster cells, rat cells, mouse cells, insect cells, or hybridomas derived therefrom). Preferably, it may be a cell derived from a mammal, including a human.

Prokaryotes suitable for this purpose are Gram-negative or Gram-positive organisms, for example Enterobacteriaceae, for example Escherichia, for example E. E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g. Salmonella typhimurium, Serratia, e.g. For example, Serratia marcescans and Shigella, and Bacilli, for example, B. B. subtilis and B. subtilis. B. licheniformis, Pseudomonas, for example P. aeruginosa and Streptomyces. The cell of the present invention is not particularly limited as long as it is capable of expressing the vector of the present invention, preferably this. It can be coli.

As a cell of the present invention, Saccharomyces cerevisiae is the most commonly used eukaryote. However, many other genera, species and strains, but are not limited to, for example, Schizosaccharomyces pombe, Kluyveromyces host, eg K. K. lactis, K. K. fragilis (ATCC 12,424), K. K. bulgaricus (ATCC 16,045), K. Wickerami (K.wickeramii) (ATCC 24,178), K. Walt (K. waltii) (ATCC 56,500), K. Drosophilaroom (K. drosophilarum) (ATCC 36,906), K. Thermotolerans (K. thermotolerans) and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Tricot Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, e.g., Shivanomiyces oxidentalis; and filamentous fungi , For example Neurospora, Penicillium, Tolypocladium and Aspergillus hosts such as A. nidulans and A. niger It is possible.

The term 'transformation' refers to the modification of the host cell's genotype by introduction of the foreign polynucleotide, and refers to the introduction of the foreign polynucleotide into the host cell regardless of the method used for the transformation. Exogenous polynucleotides introduced into a host cell may remain integrated or unintegrated into the genome of the host cell, and the present invention includes both.

A recombinant expression vector capable of expressing an antibody or fragment thereof that specifically binds to the KRS N-terminal region according to the present invention is a method known in the art, for example, but not limited to, transient transfection (transient transfection) ), Microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene- Introduced into cells to produce antibodies or fragments thereof by known methods for introducing nucleic acids into cells, such as mediated transfection (polybrene-mediated transfection), electroporation, gene guns, etc. Can be transformed.

The present invention, (a) transforming a host cell with the recombinant expression vector;

(b) culturing the transformed host cell to produce an antibody or fragment thereof; And

(c) lysyl-tRNA synthetase exposed to the outer cell membrane, comprising the step of obtaining an antibody or a fragment produced in a host cell ( KRS , Lysyl - tRNA synthetase ) It provides a method for producing an antibody or fragment thereof that specifically binds to the N-terminus.

Step (a) is a step of transforming a host cell into a recombinant expression vector operably linked to a polynucleotide encoding the antibody or a fragment thereof to produce an antibody or fragment thereof according to the present invention.

A person skilled in the art can perform this step by selecting an appropriate transformation method as described above according to the selected host cell and recombinant expression vector. Recombinant expression vectors containing heavy chain and light chain base sequences can be co-transformed into the same host cell so that heavy and light chains can be expressed in one cell, and separate recombinant expression vectors containing heavy chain and light chain base sequences are separated from each other. It can be transformed into the host cell of the heavy chain and light chain can be expressed separately.

Step (b) is a step of culturing the transformed host cell to produce a polypeptide of the heavy chain, light chain or antibody fragment of the antibody according to the present invention from a recombinant expression vector introduced into the host cell.

The medium composition for culturing the host cell, culture conditions, culture time, etc. can be appropriately selected according to methods commonly used in the art. The antibody molecule produced in the host cell can be accumulated in the cytoplasm of the cell, secreted into the cell or culture medium by an appropriate signal sequence, or targeted by periplasm. In addition, it is preferable that the antibody according to the present invention refolds the protein using a method known in the art to maintain the binding specificity to the KRS N-terminus and has a functional conformation. In addition, when producing an antibody in the form of IgG, the heavy and light chains can be expressed in separate cells, and in separate steps, the heavy and light chains can be contacted to form a complete antibody, and the heavy and light chains are expressed in the same cell. It is also possible to form a complete antibody inside the cell.

Step (c) is a step of obtaining an antibody or a fragment produced in a host cell.

Considering the characteristics of the antibody or fragment polypeptide produced in the host cell, the characteristics of the host cell, the expression method, or whether the target of the polypeptide, etc., a person skilled in the art can appropriately select and control the method of obtaining. For example, the antibody secreted into the culture medium or a fragment thereof can be recovered by obtaining a medium in which the host cell is cultured, and centrifuging to remove impurities. If necessary, cells may be lysed within a range that does not affect the functional structure of the antibody or fragment thereof in order to release and recover the antibody present in a specific organelle or cytoplasm in the cell outside the cell. In addition, the obtained antibody may be further subjected to a process of further removing and concentrating impurities through methods such as chromatography, filtration by a filter, and dialysis.

The polypeptide of the production (production) method of the present invention may be the antibody of the present invention or a fragment itself, and may be an amino acid sequence other than the antibody of the present invention or a fragment thereof, which is additionally bound. In this case, it can be removed from the antibody or fragment thereof of the present invention using methods well known to those skilled in the art.

The antibody or fragment thereof of the present invention specifically binds to the KRS N-terminus, and thus is useful for diagnostic analysis to detect and quantify, for example, KRS protein in a specific cell, tissue, or serum. In particular, it is possible to specifically detect the KRS N-terminal exposed to the outer cell membrane without lysing the cells. Therefore, the present invention comprises the steps of contacting the antibody or fragment thereof with a sample; And detecting the antibody or fragment thereof, a specific detection method of the N-terminus of Lysyl-tRNA synthetase (KRS) exposed to the outer cell membrane.

The detection method of the present invention is the presence or absence of KRS (or a KRS N-terminal peptide exposed to the outer cell membrane) using the antibody or fragment thereof according to the present invention, before contacting the antibody or fragment thereof according to the present invention with a sample. It may include the step of preparing a sample for measuring the concentration (step (1)).

Those skilled in the art can appropriately select a known method for detecting a protein using an antibody, and prepare a sample suitable for the selected method. In addition, the sample may be a cell or tissue, blood, whole blood, serum, plasma, saliva, cerebrospinal fluid, etc. obtained from a biopsy taken from a subject wishing to diagnose cancer (especially breast cancer or lung cancer) or cancer metastasis. The method for detecting a protein using the antibody is not limited to, for example, Western blot, immunoblot, dot blot, immunohistochemistry, enzyme immunoassay (ELISA), radioimmunoassay , Competitive binding analysis, and immunoprecipitation. For example, in order to perform Western blot, a buffer suitable for electrophoresis may be added to a sample or a cell lysate to prepare it for boiling, and for immunohistochemical staining, cells or tissue sections are fixed and blocked. Pre-treatment such as blocking can be performed.

Next according to the invention The step of contacting the antibody or a fragment thereof with the sample prepared in the above-described step (step (2)) is performed.

The antibody according to the present invention is an antibody or a fragment thereof that specifically binds to the KRS N-terminus having the above-described CDR, or VH and VL configurations, and the specific types and sequence configurations are as described above.

The antibody or fragment thereof can be generally labeled with a detectable moiety for its 'detection'. See, eg, Current Protocols in Immunology, Volumes 1 and 2, 1991, Coligen et al., Ed. Wiley-Interscience, New York, N. Y., Pubs, can be labeled with radioactive isotopes or fluorescent labels. Or various enzyme-substrate labels are available, examples of such enzymatic labels are luciferases, luciferin, 2,3-dihydro, such as Drosophila luciferase and bacterial luciferase (US Pat. No. 4,737,456). Peroxidase such as thalazinediones, malate dehydrogenase, urase, horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide Oxidase (eg glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (eg uricase and xanthine oxidase), lactoperoxidase, microper And oxidase. Techniques for conjugating enzymes to antibodies are described, for example, in O'Sullivan et al., 1981, Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (J. Langone & H. Van Vunakis, eds.), Academic press, N. Y., 73: 147-166. Labels can be conjugated to antibodies directly or indirectly using a variety of known techniques. For example, the antibody can be conjugated to biotin and any markers belonging to the three broad categories mentioned above can be conjugated to avidin, or vice versa. Biotin selectively binds to avidin, so this label can be conjugated to the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label to the antibody, the antibody can be conjugated with a small hapten (eg, digoxin) and one of the different types of labels mentioned above is anti- Can be conjugated to hapten antibodies (eg, anti-dioxine antibodies). Thus, indirect conjugation of the label to the antibody can be achieved.

In the present invention, "contacting (contacting)" is used in its general sense, and means that two or more substances are mixed, bonded, or brought into contact with each other. The contacting can be carried out in vitro or on other containers, and can also be performed in situ, in vivo, in an individual, in tissue, in cells.

Next, the step of detecting the antibody according to the present invention or a fragment thereof (step (3)) in the sample after step (2) is performed.

The 'detection' targets the complex of the antibody or fragment and antigen according to the present invention formed in a sample, and the presence or absence of a peptide (or a protein containing the same, for example, KRS) of the KRS N-terminal region It means detecting or measuring the level of the peptide (including both qualitative and quantitative measurements). Therefore, after performing the step (2) and before the detection step (step (3)) described below, a step of removing the extra antibody or fragments not forming a complex with the KRS N-terminal region may be further included.

When the antibody or fragment thereof used in the step (2) described above contains a detectable moiety such as fluorescently, radioactive isotope, enzyme, or the like, and is directly labeled with a method known in the art for detecting the moiety. Accordingly, detection can be performed. In one example, radioactivity may be measured, for example, by scintillation counting, and fluorescence may be quantified using a fluorimeter.

In addition, when the antibody used in step (2) described above or a fragment thereof does not contain the above-described detection moiety as such, using a secondary antibody labeled with fluorescence, radioactivity, enzymes, etc., as is known in the art. It can be detected indirectly. The secondary antibody binds to an antibody or fragment thereof (primary antibody) according to the present invention.

Through recent research, human KRS (lysyl-tRNA synthetase), which is generally present in the cytosol, is translocated to the plasma membrane (cell membrane) and 67LR (67-kDa laminin receptor) present in the plasma membrane It has been demonstrated that it promotes the migration of tumor (or cancer) cells by interacting with) and affects metastasis of cancer (Dae Gyu Kim et al., Chemical inhibition of prometastatic lysyl-tRNA synthetase-laminin receptor interaction) , Nat Chem Biol. 2014 Jan; 10 (1): 29-34.). At this time, the KRS terminal extension (N-ext) region was found to be essential for KRS to be translocated to the cell membrane. It has also been found that in relation to cancer metastasis, the KRS N-ext region is specifically involved in their binding in the interaction with 67LR.

Antibodies and fragments thereof according to the present invention have excellent specific binding ability to the KRS N-ext region and inhibit the metastasis of cancer by inhibiting the binding (interaction) with the laminin receptor as it is actually bound to the KRS N-ext region. The ability to do is excellent. This is well illustrated in the specification examples of the present invention. In the example of the present specification, as a result of administering the antibody according to the present invention to a cancer metastasis in vivo cancer metastasis mouse model, it was confirmed that it exhibits excellent cancer metastasis efficacy in a concentration-dependent manner. In particular, it has been shown that the cancer metastasis inhibitory effect is very excellent even when compared to the YH16899 compound, which is known to inhibit cancer metastasis by inhibiting the interaction of the laminin receptor (67LR) with KRS.

Therefore, the present invention provides a pharmaceutical composition for inhibiting cancer metastasis, and a composition for diagnosing cancer, comprising the antibody of the present invention or a fragment thereof as an active ingredient.

The type of cancer is not particularly limited as long as it is known as a malignant tumor in the art, but breast cancer, colorectal cancer, lung cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular Melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, parathyroid Renal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or urinary tract cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem nerve It may be selected from the group consisting of gliomas and pituitary adenoma. Preferably, in the present invention, the cancer may be breast cancer or lung cancer.

The pharmaceutical composition according to the present invention may comprise the antibody or fragment thereof of the present invention alone, or may further include one or more pharmaceutically acceptable carriers. "Pharmaceutically acceptable" in the above is a non-toxic composition that is physiologically acceptable and does not inhibit the action of the active ingredient when administered to humans and does not normally cause allergic or similar reactions such as gastrointestinal disorders, dizziness, or the like. Says

In the pharmaceutical composition according to the present invention, the antibody or fragment thereof may be administered in various dosage forms, oral and parenteral, during clinical administration. When formulated, fillers, bulking agents, binders, wetting agents, disintegrants commonly used , It may be prepared using a diluent or excipient, such as a surfactant. Solid preparations for oral administration include tablets, patients, powders, granules, capsules, troches, and the like, and the solid preparations may be added to one or more aryl derivatives of Formula 1 of the present invention, or pharmaceutically acceptable salts thereof. It may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose or gelatin. In addition, lubricants such as magnesium stearate, talc, etc. may be used in addition to simple excipients. Liquid preparations for oral administration include suspending agents, intravenous solutions, emulsions or syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, are included. Can be.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspension solutions, emulsions, lyophilized preparations, and suppositories. The therapeutic composition of the present invention may be any physiologically acceptable carrier, excipient or stabilizer (Remington: The Science and Practice of Pharmacy, l9th Edition, Alfonso, R., ed, Mack Publishing Co. (Easton, PA: 1995) )) Can be prepared in the form of a freeze-dried cake or an aqueous solution for mixing and storing the antibody having the desired purity. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations used, and buffer solutions such as phosphoric acid, citric acid and other organic acids; Antioxidants including ascorbic acid; Low molecular weight (less than about 10 residues) polypeptides; Proteins such as serum albumin, gelatin or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, arginine or lysine; Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; Chelating agents, such as EDTA; Sugar alcohols such as mannitol or sorbitol; Salt-forming counterions such as sodium; And / or nonionic surfactants such as tween, pluronics or polyethylene glycol (PEG).

The antibody of the present invention can be administered in a pharmaceutically effective amount to an individual suffering from cancer. In the above, “a pharmaceutically effective amount” refers to an amount that exhibits a higher response than a negative control, preferably an amount sufficient to treat cancer or an amount sufficient to inhibit cancer metastasis. The total effective amount of the antibody or fragment thereof of the present invention can be administered to a patient in a single dose, and can be administered by a fractionated treatment protocol that is administered for a long time in multiple doses. have. The dose of the antibody or fragment thereof of the present invention to the human body is generally 0.01-100 mg / kg / week, preferably 0.1-20 mg / kg / week, more preferably 5-10 mg / kg / It can be week. However, the dose of the antibody or fragment thereof of the present invention is considered to the patient considering various factors such as the patient's age, weight, health status, sex, disease severity, diet and excretion rate, as well as the route and frequency of administration of the pharmaceutical composition. Since the effective dosage for a patient is determined, those who have ordinary skill in the art in consideration of this point will be able to determine an appropriate effective dosage for the specific use of the antibody or fragment thereof of the present invention as a cancer metastasis inhibitor. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, route of administration and method of administration as long as it shows the effect of the present invention.

The route of administration of the compositions of the invention can be administered or injected by known methods of antibody administration, e.g. intravenous, intraperitoneal, intracranial, subcutaneous, intramuscular, intraocular, intraarterial, cerebrospinal, or intralesional routes, or Injection or infusion by the described sustained release system. In one example, the antibodies of the invention can be administered systemically or locally.

The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, hormonal therapy, chemotherapy, and biological response modifiers for the prevention or treatment of cancer.

Diagnosis and prognostic evaluation of cancer (or cancer metastasis) according to the present invention can be performed by detecting KRS protein (in particular, the KRS N-terminal region exposed to the outer membrane) in a biological sample.

In the present invention, the term 'diagnosis' means to identify the presence or characteristic of a pathological condition. In the present invention, the diagnosis is to confirm whether cancer or / and cancer metastasis has occurred or not, and the likelihood (risk).

“Detection” is as described above, and the biological sample includes blood or other liquid samples of biological origin, biopsy sample, solid tissue sample such as tissue culture, or cells derived therefrom. More specifically, for example, but not limited to, tissue, extract, cell lysate, whole blood, plasma, serum, saliva, ocular fluid, cerebrospinal fluid, sweat, urine, milk, ascites fluid, synovial fluid, peritoneal fluid, and the like. The sample can be obtained from an animal, preferably a mammal, most preferably a human. The sample can be pretreated prior to use for detection. For example, it may include filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like. In addition, nucleic acids and proteins can be separated from the sample and used for detection.

The antibody or fragment thereof according to the present invention may be provided as a diagnostic kit, and the kit is not particularly limited as long as it is known in the art as an analysis kit for providing an antibody or a peptide having a specific binding domain as a component. Examples include Western blot, ELISA, radioimmunoassay, radioimmunoassay, oakteroni immunodiffusion, locator immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS or kit for protein chip.

The antibody or fragment thereof of the present invention can be used in a packaged combination of reagents in a predetermined amount with instructions for use in the kit, that is, a diagnostic kit for performing diagnostic analysis. When the antibody is labeled with an enzyme, the kit may contain a cofactor required by the enzyme as a substrate precursor that provides a substrate and a chromophore or fluorophore. In addition, other additives may be included, such as stabilizers, buffers (eg, blocking buffer or lysis buffer), and the like. The relative amounts of various reagents can be varied widely to provide concentrations in solution of reagents that fully optimize the sensitivity of the assay. Reagents can be provided as generally lyophilized, dry powders that contain excipients that, upon dissolution, will provide a reagent solution with an appropriate concentration.

Antibodies or fragments thereof according to the present invention have a specific CDR (complementarity determining region) sequence described herein and have a very good specific binding ability to the KRS N-terminal region exposed to the outer cell membrane. In addition, since it is specifically targeted to the KRS N-terminal region in vivo, the effect of suppressing cancer metastasis by suppressing the interaction of the laminin receptor with the KRS N-terminal region is excellent.

Figure 1 shows the results of selecting a scFv phage clone that binds to the KRS full-length sequence or KRS N-terminal fragment through Western blot (WB).
Figure 2 shows the results of selecting the scFv phage clone that binds to the KRS full-length sequence or KRS N-terminal fragment through immunoprecipitation (IP).
3A is a result of confirming whether scFv clones of N3, N5, N7, and N9 are bound to the exposed region, respectively, by producing cells exposed to the KRS-N terminal region on the cell membrane through myc-KRS T52D (Active mutant) expression. (KRS: means myc-KRS T52D).
FIG. 3B shows that the KRS-N terminal region is not exposed to the cell membrane in the inactive mutant (myc-KRS T52A) or WT-KRS (laminin untreated) expression group, and thus detection is performed by scFv clones of N3, N5, N7, and N9 It shows that there is no signal.
Figure 3C is a myc-labeled WT-KRS, T52D, T52A cells transformed with laminin and then scFv stained, WT-KRS laminin-induced membrane localization and T52D mutant are stained at similar positions , The transformed myc was also stained at the same location, but it was not stained at T52A (scFv: green, myc: red).
Figure 4 is a full length KRS (denoted by F, SEQ ID NO: 76), N-terminal 1-71 amino acid deletion KRS fragment (denoted by 1), and N-terminal 1-200 amino acid KRS fragment consisting of Using (denoted as 2), the result of Western blot confirming whether scFv clones of N3, N5, N7 and N9 specifically bind to KRS N-terminus.
Figure 5A shows the results of confirming the binding ability of N3 IgG and N5 IgG to KRS by Western blot among antibodies of the present invention.
Figure 5B shows the results of confirming the binding ability of N3 IgG and N5 IgG to KRS by immunoprecipitation, among the antibodies of the present invention.
6A shows the result of quantitatively confirming the binding force of N3 IgG to the KRS N-terminus through the SPR method.
6B shows the result of quantitatively confirming the binding force of N5 IgG to the KRS N-terminus through the SPR method.
Figure 7A is treated with laminin on cells transformed to express WT-KRS so that the N-terminal region of KRS is exposed to the outer cell membrane, and then the antibody N3 IgG of the present invention is treated to immunize binding to the exposed region The results confirmed by the fluorescent dyeing method are shown.
Figure 7B is T52D-KRS (active mutant, myc tagged KRS) through the expression of the cell membrane to produce a cell exposed to the KRS-N terminal region, the antibody of the present invention N3 IgG treatment to bind to the exposed region immunofluorescence The results confirmed by the staining method are shown. In addition, in the experiment of imparting permeability to the cell membrane, it was confirmed that the antibody of the present invention was able to enter the cell and bind to the KRS protein present inside the cell.
Figure 7C is treated with laminin on cells transformed to express WT-KRS so that the N-terminal region of KRS is exposed to the outer cell membrane, and then the antibody N5 IgG of the present invention is treated to immunize binding to the exposed region The results confirmed by the fluorescent staining method are shown (N5 IgG: green).
8 shows the results of confirming that the antibody N3 IgG of the present invention has no cytotoxicity by the MTT assay method.
9A shows the results confirming that cell migration is inhibited by the antibody N3 IgG treatment of the present invention.
9B shows the results confirming that the antibody N3 IgG of the present invention significantly inhibits cell migration depending on concentration.
FIG. 10 schematically shows the production of a mouse cancer metastasis model and the schedule of administration of a therapeutic agent (antibody or YH16899), and an experimental schedule for observing lung metastasis from the mouse model in an experiment using an in vivo cancer metastasis model.
Figure 11 shows a mouse lung sample, which can be confirmed that the lung metastasis of cancer is significantly suppressed by administration of the antibody N3 IgG of the present invention in the cancer metastasis model in vivo . The progress and severity of cancer metastasis can be evaluated by the number and status of nodules in the lung sample.
Figure 12 shows the results of confirming that the production of lung nodules is significantly inhibited (ie, lung metastasis of cancer is significantly suppressed) compared to the control group by administration of the antibody N3 IgG of the present invention in a cancer metastasis model in vivo .
Figure 13 shows the control and the lung tissue in the control group of N3 IgG in contrast, in the control group, it was confirmed that the laminin receptor was significantly expressed in the metastasized nodular region compared to the antibody treatment group of the present invention.
FIG. 14 shows lung samples from the control group, the YH16899-treated group, and the N3 IgG-treated group, respectively, and shows a result in which the generation of pulmonary nodules was significantly suppressed compared to the control group in the YH16899-treated group and the N3 IgG-treated group.
FIG. 15 shows the lung metastasis suppression efficiency (efficiency of inhibiting lung nodule production) according to the treatment concentration of the cancer metastasis inhibitor materials in the YH16899 treatment group and the N3 IgG treatment group.
Figure 16 shows the results of quantitatively confirming the binding force to the human (human, h) KRS N-terminal peptide fragments (F1, F2, F3, F4, F5) of N3 IgG through the SPR method (gray bar at the bottom of the sequence) Indicates the binding force of the N3 antibody to the corresponding region (F1 to F5), and the darker the color of the bar, the higher the binding strength.
FIG. 17 quantitatively confirms the binding capacity of N3 terminal peptide fragments (F1, F3, F5) of human (h), mouse (mouse, m), and rat (r, r) KRS N3 IgG through the SPR method. The results (the gray bars at the bottom of the sequence indicate the binding power of the N3 antibody to the corresponding regions (F1 to F5), indicating that the darker the color of the bar, the higher the binding strength).

Hereinafter, the present invention will be described in detail.

However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

<Example 1>

scFv library screening

<1-1> scFv phage screening_ 1st

Among the KRS full-length sequences (SEQ ID NO: 76), to screen for scFvs that specifically bind to the KRS N-terminal (SEQ ID NO: 75) region exposed to the outer cell membrane when transferred to the cell membrane by laminin signal, labeled with HA tag A phage display panning experiment was conducted using scFv phage library derived from human B cells. The scFv display phage library (Library size: app.7.6 x 10 ^9, Library produced by prof.Hyunbo Shim) used in this experiment is described in Korean Patent Registration No. 10-0961392. As shown in Table 1 below, KRS fragments of different specific regions of the KRS full-length sequence and the N-terminal position were used as antigenic proteins for phage display panning experiments.

To the Immuno-tube containing 1 ml of 1X PBS solution, 1-10 μg of antigen protein was added and reacted at 37 ° C and 200 rpm for 1 hour to coat the antigen on the inner surface of the tube. The antigen solution was decanted and washed once with tap water to remove uncoated antigen. To prevent non-specific binding between the antigen protein and the phage, the immuno-tube and the scFv library were reacted for 1 hour at room temperature with 1X PBST (PBS containing 0.05% tween20) containing 3% skim milk, respectively. After removing the skim milk from the immuno-tube, the scFv library was added and reacted at 37 ° C and 150 rpm for 1 hour to bind the scFv phage to the antigen. After the scFv phage was reacted in a tube, the scFv phage that was not bound was removed by washing 2 to 5 times with 1X PBST.

The scFv phage specifically bound to each KRS antigen was separated within 10 minutes by adding 1 ml of triethylamine (100 mM) at room temperature, and neutralized with Tris (1M, pH 7.4). The filtered phage scFv was added to ER2537 E. coli cultured with OD <1 and infected while incubating at 37 ° C and 120 rpm for 1 hour and 30 minutes. E.coli infected with phage was centrifuged to remove some of the culture supernatant and redispersed to apply to an agarose plate with a diameter of 15 cm containing ampicillin and glucose (2%). The next day, 5 ml SB medium was applied to obtain all the cells grown on the plate, glycerol (50%) was added to 0.5 times the total volume, mixed, and 1 ml aliquoted and stored at -80 ° C (scFv panning stock). 20 μl of the prepared stock was inoculated in a 20 ml SB solution, cultured, and prepared as a scFv phage library (1 ml) for next step phage panning using a helper phage. The above process for separating phage expressing scFv specific for the antigen was repeated 2-3 times.

<1-2> Screening of binding-specific scFv antibody through ELISA-2nd screening

Whether the scFv expressed by the phage selected in Example <1-1> binds to the aforementioned KRS full-length or N-terminal fragments was examined by conducting an indirect ELISA.

The scFv result obtained by repeating Panning three times was diluted and applied to agarose plates having a diameter of 10 cm, and the next day, each colony was selected and cultured in a 96 well plate containing 200 μl of SB medium. It was confirmed that the overall growth was good, IPTG (1 mM) was added, and cultured at 30 ° C. for 16 hours to induce production of scFv. The next day, 96-well plates were centrifuged to separate only cells, and then the cells were lysed using a TES solution and centrifuged again to separate only the supernatant. The obtained supernatant was subjected to indirect ELISA to select scFvs that specifically bind to the antigen. The KRS full-length or N-terminal fragment antigen was coated with each plate, and the culture supernatant containing scFv was reacted, followed by reaction with an anti-HA-HRP antibody (Roche Applied Science) as a secondary antibody. After the color reaction using Tetramethylbenzimidine (TMB, Thermo scientific), the color reaction was stopped using H ₂ SO ₄ (1M) and absorbance was measured at 450 nm using an ELISA reader. Control (blank) and the above-described antigen (Ag) were simultaneously performed on ELISA to select only colonies with positive values. A total of 1920 colonies were performed on ELISA, and 93 were selected as positive (see Table 1).

Antigen (KRS fragment) Full 1-20 13-36 31-46 1-29 5-34 10-38 15-42 24-49 Total Panning Outcomes 288 384 384 192 192 192 96 96 96 1920 HITS 51 8 20 2 One One 0 8 2 93

<1-3> Sequencing

In Example <1-2>, the sequence was analyzed to filter out the same Ab having the CDR sequence overlapping among 93 colonies selected through ELISA screening. Specifically, sequencing was performed in the following manner: E. coli containing scFv clone was cultured, and then phagemid was obtained by miniprep. This was confirmed using Omp primer (Hye young Yang, et. Al., 2009, Mol. Cells 27, 225-235). The sequence obtained in this way was confirmed using the Bioedit program to determine the sequence of the phagemid CDR region. Among them, clones having duplicate CDR sequences were removed, and scFv clones of independent CDR sequences were identified.

Antigen (KRS fragment) Full 1-20 13-36 31-46 1-29 5-34 10-38 15-42 24-49 Total HITS 51 8 20 2 One One 0 8 2 93 Different Clones 10 8 11 One One One 0 4 2 38

As a result of selecting antibodies of overlapping CDR sequences through sequencing, as shown in Table 2 above, there were 38 scFv clones of different CDR sequences.

<1-4> Binding specific scFv antibody screening through western blot_3rd screening

For 38 scFv clones isolated in Example <1-3>, it was confirmed by western blot whether they specifically bind to KRS.

The scFv-positive single colony clone was cultured in 5 ml of SB medium containing kanamycin (Bactotrytone 30 g, yeast extract 20 g, MOPS buffer 10 g / L) to start seed culture, overnight culture, and then transferred to 500 ml kanamycin-containing SB medium for OD 600 = 0.5 When IPTG was added to 1 mM and cultured overnight at 30 degrees, scFv protein was expressed in E. coli periplasm. The next day, the E. coli obtained by centrifugation was suspended in 1X TES buffer (50 mM Tris, 1 mM EDTA, 20% Sucrose, pH 8.0), mixed with 0.2 X TES 1.5 times, mixed and centrifuged to take a supernatant by taking a supernatant. Was extracted.

The final 5 mM MgSO ₄ was added to the scFv antibody extracted from Periplasm and mixed with Ni-NTA bead previously equilibrated in PBS and stirred in refrigeration for 1 hour to bind to Ni-NTA bead, followed by affinity chromatography. Unwashed protein was sufficiently washed with PBS. After washing more thoroughly with a buffer containing 5 mM Imidazole, the bound scFv antibody was eluted using 200 mM Imidazole buffer. The eluted antibody was dialyzed to confirm the purity through electrophoresis, and the protein was quantified using the BCA method to record the purified antibody amount, and then dispensed a certain amount and stored frozen.

As described above, the scFv antibody extracted from periplasm was used to confirm whether the scFv antibody binds to the KRS full-length or each KRS N-terminal fragment using a Western blot technique. After electrophoresis of 30 ug of HCT116 cell lysate through SDS PAGE, transferred to PVDF membrane, blocked with 3% skim milk, and then added with 1.0 μg / µl of the extracted scFv antibody for binding for 1 hour. The unbound scFv antibody was removed, and for detection, the HF (horseradish peroxidase) -linked Anti-HA secondary antibody was reacted with the scFv bound to the antigen, and then the ECL reagent was used as a substrate to excite the film in the dark. The photosensitive band was compared with the standard molecular markers to identify the KRS full length and the band corresponding to the size of each fragment.

The Western blot was performed to remove scFv clones that resulted in too weak band (faint band) and non-specific band (double band). There were 13 scFv clones selected in this way, and these results are shown in FIG. 1 .

<1-5> Binding specificity through immunoprecipitation scFv  Antibody screening-4th screening

To confirm whether the scFv clone selected in Example <1-4> actually binds to native KRS, immunoprecipitation was performed. The purified scFv clone was reacted with HCT116 cell lysate and Ag-Ab binding, and immunoprecipitation was performed using the HA-tag of scFv.

Specifically, HCT116 cells were lysed with 20 mM Tris-HCl buffer (pH 7.4, lysis buffer) containing 150 mM NaCl, 0.5% Triton X-100, 0.1% SDS and protease inhibitor. 5 μg of each scFv was added to 500 μg of HCT116 cell lysate, followed by incubation at 4 ° C. overnight. 30 μl of anti-HA agarose bead was added and incubated at 4 ° C. for 4 hours. The supernatant was removed by centrifugation. The precipitate thus obtained was dissolved in SDS-sample buffer and the process of boiling for 7 minutes was repeated twice.

After the immunoprecipitation samples prepared through the above-described process are electrophoresed through SDS PAGE, transferred to PVDF membrane and blocked with 3% skim milk, KRS polyclonal antibody (rabbit, Neomics, Co. Ltd. # NMS-01- 0005) The antibody was added and bound for 1 hour. The unbound antibody was removed and reacted by adding an anti-rabbit secondary antibody (ThermoFisher Scientific, # 31460). After reacting the secondary antibody, the film was photosensitive in the dark using ECL reagent as a substrate. The photosensitive band was compared with the standard molecular markers to identify the KRS full length and the band corresponding to the size of each fragment.

There were 12 scFv clones selected in this way, and these results are shown in FIG. 2 .

<1-6> Screening of binding specific scFv antibody through immunofluorescence method_5th screening

In order to confirm whether the scFv clone selected in Example <1-5> binds to the KRS N-terminal region exposed to the actual cell membrane, immunofluorescence was performed. At this time, KRS-T52D mutant (active mutant) was used to create a phenomenon in which the KRS was exposed to the membrane. Specifically, after dispensing A549 cells on a glass coverslip (1 x 10 ⁵ cells, based on a 12 well plate), after 24 hours, myc-KRS WT / myc-KRS T52D (Active mutant) / myc-KRS T52A (Inactive mutant) Overexpressed Thereafter, the cells were cultured for 24 hours at 37 ° C using a serum-free medium (RPMI 1640 media) for 24 hours. Thereafter, 10 μg / ml laminin (L2020; Sigma) was treated and left at 37 ° C. for 1 hour to prepare a sample. Each myc-KRS T52D (Active mutant) and myc-KRS T52A (Inactive mutant) vectors are site-directed mutagenesis (QuikChange II Site-Directed Mutagenesis kit, Agilent, # 200523) using pcDNA3-myc-KRS WT vector as a backbone. It was produced through.

The prepared sample was washed with PBS (4 ° C.), fixed with 4% paraformaldehyde for 10 minutes, and then washed. CAS block was performed for 10 minutes, and the scFv and Myc antibodies were treated for 2 hours. The unbound antibodies were then removed and reacted with secondary antibodies for 1 hour in the dark. DAPI staining was performed for 10 minutes to mount.

As shown in Fig. 3 , a total of four scFv clones (N3, N5, N7, and N9) that bind to only the cells of the active mutant without binding to Inactive mutant (T52A) and WT-KRS (laminin untreated) were selected. . In addition, it was confirmed that when scFv was stained after laminin treatment to induce membrane localization of WT-KRS, it was confirmed to be stained at a position similar to T52D mutant.

It was confirmed that these clones were specifically bound at the cell tip.

<1-7> Confirmation of specific binding ability to KRS N-terminal

To confirm whether the final selected scFv clones (N3, N5, N7, and N9) through Example <1-6> actually bind to KRS N-terminus, full length KRS (denoted by F, SEQ ID NO: 76) , Western blot was performed using the KRS fragment (labeled 1) with the N-terminal 1-71th amino acid deleted, and the KRS fragment (labeled 2) with the N-terminal 1-200th amino acid.

 Using the polynucleotides encoding the full length KRS and KRS fragments, A549 cells were transformed in the same manner as described in Example <1-6>. After that, the cells were lysed, and Western blot was performed in the same manner as described in Example <1-4> above.

As a result, as shown in Figure 4 , all of the selected scFv colne (N3, N5, N7, and N9) showed no band in fragment 1 without N-terminus 1 to 72 amino acid, and a fragment with KRS 1 to 200 amino acid It was confirmed that the band was visible only at As a result, it was confirmed that scFv clones of N3, N5, N7, and N9 specifically bind to KRS N-terminus.

<1-8> Sequence analysis of KRS N-terminal binding-specific scFv clone

The CDR constructs and VH and VL sequence analysis were performed on the scFv clones (N3, N5, N7, and N9) finally selected through Example <1-6>. Sequencing was performed in the same manner as in Example <1-3> described above.

As a result of sequencing, the N3 scFv consisted of the amino acid sequence represented by SEQ ID NO: 67, and the sequence included the linker sequence of SEQ ID NO: 65 in the middle. Accordingly, the VH of N3 consisted of the amino acid sequence represented by SEQ ID NO: 49, and the VL of N3 consisted of the amino acid sequence represented by SEQ ID NO: 51. As a result of analyzing the respective CDR sequences included in the VH and VL of N3, the VH of N3 shows heavy chain CDR1 represented by SEQ ID NO: 1, heavy chain CDR2 represented by SEQ ID NO: 3 and heavy chain CDR3 represented by SEQ ID NO: 5 The VL of N3 contained light chain CDR1 represented by SEQ ID NO: 7, light chain CDR2 represented by SEQ ID NO: 9, and light chain CDR3 represented by SEQ ID NO: 11.

The N5 scFv consisted of the amino acid sequence represented by SEQ ID NO: 69, and the sequence included the linker sequence of SEQ ID NO: 65 in the middle. Accordingly, the VH of N5 consisted of the amino acid sequence represented by SEQ ID NO: 53, and the VL of N5 consisted of the amino acid sequence represented by SEQ ID NO: 55. The VH of N5 includes heavy chain CDR1 represented by SEQ ID NO: 13, heavy chain CDR2 represented by SEQ ID NO: 15 and heavy chain CDR3 represented by SEQ ID NO: 17, and the VL of N5 is light chain CDR1 represented by SEQ ID NO: 19, SEQ ID NO: Light chain CDR2 represented by 21 and light chain CDR3 represented by SEQ ID NO: 23 were included.

The N7 scFv consisted of the amino acid sequence represented by SEQ ID NO: 71, and the sequence contained the linker sequence of SEQ ID NO: 65 in the middle. Accordingly, the VH of N7 consisted of the amino acid sequence represented by SEQ ID NO: 57, and the VL of N7 consisted of the amino acid sequence represented by SEQ ID NO: 59. The VH of N7 includes heavy chain CDR1 represented by SEQ ID NO: 25, heavy chain CDR2 represented by SEQ ID NO: 27, and heavy chain CDR3 represented by SEQ ID NO: 29, and VL of N7 is light chain CDR1 represented by SEQ ID NO: 31, SEQ ID NO: Light chain CDR2 represented by 33 and light chain CDR3 represented by SEQ ID NO: 35 were included.

The N9 scFv consisted of the amino acid sequence represented by SEQ ID NO: 73, and the sequence contained the linker sequence of SEQ ID NO: 65 in the middle. Accordingly, the VH of N9 consisted of the amino acid sequence represented by SEQ ID NO: 61, and the VL of N9 consisted of the amino acid sequence represented by SEQ ID NO: 63. The VH of N9 includes heavy chain CDR1 represented by SEQ ID NO: 37, heavy chain CDR2 represented by SEQ ID NO: 39, and heavy chain CDR3 represented by SEQ ID NO: 41, and the VL of N9 is light chain CDR 1 represented by SEQ ID NO: 43, sequence Light chain CDR2 represented by No. 45 and light chain CDR3 represented by SEQ ID NO: 47 were included.

< Example  2>

scFv  Antibody With IgG  Conversion and specific binding evaluation

<2-1> scFv  Antibody With IgG  transform

First, polynucleotides encoding scFv in the genome of clones N3, N5, N7 and N9 phage were amplified by PCR. The base sequence of the primer used to amplify the gene of the VH region of the scFv is as follows: Forward (AGA GAG TGT ACA CTC C CA GGC GGC CGA GGT GCA G, SEQ ID NO: 93), Reverse (CGC CGC TGG GCC CTT GGT GGA GGC TGA GCT CAC GGT GAC CAG, SEQ ID NO: 94) The base sequence of the primer used to amplify the gene of the VL region of scFv is as follows: Forward (AAG CGG CCG CCA CCA TGG GAT GGA GCT GTA TCA TCC TCT TCT TGG TAG CAA CAG CTA CAG GTG TAC ACT CCC AGT CTG TGC TGA CTC AG, SEQ ID NO: 95), Reverse (CGC CGC CGT ACG TAG GAC CGT CAG CTT GGT, SEQ ID NO: 96)

Each phage DNA (50 ng) as a template using the primer (10 pmol each) 95 ℃ / 3min; 95 ° C / 30sec, 60 ° C / 30sec, 72 ° C / 30sec, 30 cycles; PCR was performed under the condition of 72 ° C./5 min to amplify the VH or VL gene of N3, N5, N7 or N9 scFv. The PCR product was inserted into pcDNA3.4 vector, a vector used for IgG production, using restriction enzymes. Proteins of the heavy chain and light chain of IgG were individually expressed in separate plasmids.

The vector comprising the DNAs encoding the light and heavy chains of IgG (hereinafter referred to as N3 IgG, N5 IgG, N7 IgG, and N9 IgG) containing the variable region of scFv prepared above is co-transformed into freestyle 293F cells. The light and heavy chains were expressed together in cells. The transformed 293F cells were cultured at 37 ° C and 8% CO ₂ for 7 days to obtain a supernatant. The supernatant was filtered using a cellulose acetate membrane filter (pore size 0.22 μm, Corning) and purified using CaptivA ™ PriMAB protein A column (Repligen, USA). The obtained antibody concentration was measured using a BCA kit (Pierce, 23225), and the IgG antibody protein produced under reduced and non-reduced conditions was analyzed using a Bioanalyzer (Agilent 2100 Bioanalyzer).

<2-2> converted IgG KRS  Bond strength verification _ Western Blot  And immunoprecipitation

The binding strength of the IgGs prepared in Example <2-1> to KRS was verified by Western blot (WB) and immunoprecipitation (IP). Western blots were performed in the same manner as described in Example <1-4>, and immunoprecipitation was performed in the same manner as described in Example <1-5>.

As a result, the IgGs produced in the present invention were verified to bind to KRS, and FIG. 5 shows these results as representatives of N3 IgG and N5 IgG.

<2-3> converted IgG KRS  Verification of N-terminal specific binding force _ SPR

Quantitative binding capacity between purified antibody proteins (N3 IgG and N5 IgG) and antigen (KRS 1-207 aa) was measured using a Biacore 2000 Surface Plasmon Resonance (SPR) (GE healthcare, USA) biosensor. Antibody sequentially diluted in HES buffer solution (10 mM HEPES, pH7.4 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20) after fixing KRS to sensor chip CM5 (GE healthcare, USA) Protein (6.25 ~ 100 nM) was flowed at a rate of 30 μl / min for 3 minutes, and 1M NaCl / 20mM NaOH was flowed at a rate of 30 μl / min for 3 minutes to induce protein dissociation bound to the antigen. Mock IgG was used as a control. The specific experimental conditions are as follows;

Immobilized Antigen: KRS

Immobilized level: 185 RU

Antibody: N3 IgG, N5 IgG

Running buffer: HBS-N buffer

Regeneration: 2 M NaCl, 20 mM NaOH (flow 30 ul / min 1 min)

ka (1 / Ms) kd (1 / s) KD (M) N3 IgG 1.09E + 05 0.009055 8.34E-08 N5 IgG 3.13E + 06 0.003282 1.05E-09

Table 3 shows the movement rate constants and the equilibrium dissociation constants of N3 IgG and N5 IgG measured using Biacore 2000 SPR. Using the BIA evaluation ver.3.2 software, the affinity was obtained as the kinematic constant (ka and kd) and the equilibrium dissociation constant (KD). 6 shows the SPR graph results of N3 IgG and N5 IgG, respectively. From FIG. 6 and Table 3 , it was confirmed that the N3 IgG and N5 IgG of the present invention specifically have high binding strength to the KRS N-terminal region. There was no binding signal in the control mock IgG.

<2-4> Verification of KRS N-terminal specific binding ability of converted IgG_Immunofluorescence staining

Immunofluorescence staining was performed to confirm that the IgG produced in the present invention binds to the KRS region exposed to the actual cell membrane. Typically, using N3 IgG and N5 IgG, immunofluorescence staining was performed in the same manner as described in Example <1-6> above.

As a result, as shown in FIG. 7A , when laminin was treated to induce membrane localization of WT-KRS, it was confirmed that N3 IgG specifically bound well to the KRS N-terminal portion exposed to the outer cell membrane. As shown in FIG. 7B, in the experiment using T52D-KRS (active mutant, myc tagged KRS), the antibody of the present invention specifically well bound to the KRS N-terminal portion exposed to the outer cell membrane, and the cell membrane to the experimental cell When the permeability was imposed, the KRS protein present in the cells was detected with high sensitivity. In addition , as shown in FIG. 7C , when laminin was treated to induce membrane localization of WT-KRS, it was confirmed that N5 IgG specifically bound well to the KRS N-terminal portion exposed to the outer cell membrane.

Thus, it was confirmed that the antibodies provided in the present invention have high binding specificity to the KRS N-terminal portion exposed to the outer cell membrane.

<Example 3>

Confirmation of cancer metastasis suppression efficacy

<3-1> Cytotoxicity evaluation

 Typically, MTT assay was performed using N3 IgG. A549 cells (5,000 cells / well) were dispensed into 96-well plates and cultured. After washing the cells using serum-free medium, human mock IgG and N3 IgG were treated with concentrations of 0, 50, 100, and 500 nM (in serum free media), respectively. After incubation for 24 hours, 50 μg / well of MTT solution was added and treated for 4 hours. After removing the MTT solution, 100 ul of DMSO was treated and absorbance was measured at 570 nm.

As shown in FIG. 8 , it was confirmed that the antibody of the present invention does not exhibit cytotoxicity.

<3-2> Cell migration analysis

Cell migration is poly as described in the prior literature (Park, SG et al. Human lysyl-tRNA synthetase is secreted to trigger pro-inflammatory response, Proc. Natl. Acad. Sci. USA 102, 6356-6361 (2005)) Measured in a 24-well transwell chamber with carbonate membrane (8.0 μm pore size, Costar). The lower well was coated with 10 ug Laminin (in gelatin) in the transwell chamber and dried with UV. The A549 cells were then suspended in serum-free RPMI medium and then placed in the upper chamber at a concentration of 1 x 10 ⁵ cells per well. N3 IgG or human mock IgG (control) was treated with 100 nM or 500 nM in the chamber and incubated for 24 hours. Then, washed twice with PBS and treated with 70% MeOH (in PBS) for 30 minutes. Again washed twice with PBS and Hematoxylin solution was treated for 30 minutes. The chamber was washed three times with DW, the membrane in the chamber was cut and mounted on slide glass.

As shown in Fig. 9A, N3 IgG showed the effect of significantly inhibiting the migration of A549 cells. In addition, this cell migration inhibitory effect was shown to be concentration-dependent (see FIG. 9B).

<3-3> in vivo In cancer metastasis model, evaluation of cancer metastasis suppression efficacy

Since KRS can promote cell migration through 67LR associated with cancer metastasis, a tumor (cancer) animal model was constructed using mouse breast cancer 4T-1 cells (Korea Cell Line Bank), which metastasize well to the lung. 4 x 10 ⁴ cells in 6 7-week-old BALB / cAnCr mice 4T1 cells were injected into the fat pad of the mouse to prepare an orthotopic breast cancer animal model.

Cancer was injected into the Mammmary Fat pad 10 days later (day10), and the cancer tissue was removed from the Fat pad. After 1 day (day11), N3 IgG is administered at 3 mg intervals, 2 times a week, 2 weeks (4 times, day11, 14, 18, 21) at 10 mg / kg tail vein iv injection And control mock IgG (Thermo # 31154) was also administered at the same dose. After completion of all antibody administration schedules, one week later, 28 days after cancer injection, mice were sacrificed to obtain lung tissue. At the time of necropsy of the lung tissue, saline was injected using a syringe into the bronchus, the lungs were inflated, and then collected and stored in Bouin's solution (Sigma # HT10132) for 24 hours. Thereafter, the number of metastasis nodules metastasized from each lobe of the lung was checked through a microscope.

As shown in FIGS . 11 and 12 , in the control group, it was confirmed that many nodules were formed in the lungs due to cancer metastasis, and it was confirmed that these nodules were significantly suppressed in the N3 IgG treatment group. Figure 13 shows the control and the lung tissue in the control group of N3 IgG in contrast, in the control group, it was confirmed that the laminin receptor was significantly expressed in the metastasized nodular site compared to the antibody treatment group of the present invention.

<3-4> in vivo In cancer metastasis model, efficacy comparison with anti-cancer metastasis compound (YH16899)

In the literature of 'Dae Gyu Kim et al., (2014)', a compound called YH16899 inhibits the interaction between 67LR and KRS and is known to be effective in suppressing cancer metastasis. Thus, the cancer metastasis inhibitory effect of YH16899 and the antibody N3 IgG of the present invention was compared. The construction of an in vivo tumor model and observation of lung metastasis were performed in the same manner as in Example <3-3>. YH16899 was orally administered daily at 100 mpk. N3 IgG was injected intravenously through the tail of the mouse by concentration (1mpk, 10mpk), respectively.

As shown in FIGS . 14 and 15 , it was confirmed that the number of lung nodules was significantly reduced in the concentration-dependent manner in the N3 IgG-treated group, and compared with the YH16899 100-mpk-treated group, only N3 IgG was treated at a dose of 1 mpk. It was also confirmed that cancer metastasis was significantly suppressed.

< Example  4>

KRS  Check the binding position of the antibody

<4-1> KRS Monoclonal  Antibody human KRS  Joining position

Among the KRS antibodies prepared above, SPR (Surface Plasmon Resonance) experiment was performed as follows to confirm the position of N3 IgG binding to human KRS.

First, N3 IgG antibody was fixed to a Biacore T200 (GE Healthcare) equipped with a Series S sensor chip CM5 (GE Healthcare) using an amine coupling kit (GE Healthcare). Then, the peptides listed in Table 4 were dissolved in PBS solution at a corresponding concentration and flowed for 60 seconds. Then, PBS was flowed for 5 minutes. Then, the binding force was analyzed with Biacore T200 Evaluation software v2.0 (GE Healthcare).

Peptide information designation Sequence information Species MW Sequence number F1 (1-29) MAAVQAAEVKVDGSEPKLSKNELKRRLKA H 3168 98 F2 (5-34) QAAEVKVDGSEPKLSKNELKRRLKAEKKVA H 3351 99 F3 (10-38) KVDGSEPKLSKNELKRRLKAEKKVAEKEA H 3310 100 F4 (15-42) EPKLSKNELKRRLKAEKKVAEKEAKQKE H 3337 101 F5 (24-49) KRRLKAEKKVAEKEAKQKELSEKQLS H 3084 102 mF1 (1-28) MATLQESEVKVDGEQKLSKNELKRRLKA M 3230 103 mF2 (3-34) QESEVKVDGEQKLSKNELKRRLKAEKKLA M 3383 104 mF3 (10-37) KVDGEQKLSKNELKRRLKAEKKLAEKEA M 3268 105 mF4 (15-41) QKLSKNELKRRLKAEKKLAEKEAKQKE M 3253 106 mF5 (24-48) RRLKAEKKLAEKEAKQKELSEKQLN M 2997 107 rF1 (1-28) MATLREGEVKLDGEPKLSKNELKRRLKA R 3211 108 rF2 (3-34) REGEVKLDGEPKLSKNELKRRLKAEKKLA R 3364 109 rF3 (10-37) KLDGEPKLSKNELKRRLKAEKKLAEKEA R 3251 110 rF4 (15-41) PKLSKNELKRRLKAEKKLAEKEAKQKE R 3222 111 rF5 (24-48) RRLKAEKKLAEKEAKQKELSEKQLN R 2997 112

As a result, as shown in FIG. 16, the epitopes F1, F2, F3, and F4 showed that the N3 IgG antibody binds, whereas the F5 showed that the N3 IgG antibody did not bind. In addition, in the case of epitope F4, the binding strength was found to be the strongest, and in the order of F3, F2, and F1, the binding strength was found to be strong.

Through this, it was confirmed that the position where the N3 IgG antibody mainly binds is the 15-29 amino acid region of the KRS N-terminal region.

<4-2> KRS Monoclonal  Cross activity between different species of antibodies

In the above example, to confirm whether the KRS antibody N3 IgG binds to human KRS, and to confirm whether the N3 IgG shows cross reactivity with other species of mouse (m) and rat (r), SPR (Surface) as follows: Plasmon Resonance) experiment was conducted.

The N3 IgG antibody was fixed to the chip using an amine coupling kit (GE Healthcare) in the same manner as the experimental method described in Example 4-1, and the peptides listed in Table 4 were dissolved in PBS solution at a corresponding concentration 60 It was allowed to flow for seconds, followed by PBS for 5 minutes. Then, the binding force was analyzed with Biacore T200 Evaluation software v2.0 (GE Healthcare).

As a result, as shown in FIG. 17, it was shown that the N3 IgG antibody binds to epitopes F1, F2, F3, F4 of human (h), mouse (m), and rat (r), and not F5. In addition, it was found that epitope F3 has a stronger binding force than F1 in humans, mice, and rats (F2, F4 data not shown).

Through this, it was confirmed that the N3 IgG antibody can cross-react between species.

As described above, the antibodies or fragments thereof according to the present invention have a specific CDR (complementarity determining region) sequence described herein and have a very good specific binding ability to the KRS N-terminal region exposed to the outer cell membrane, It is available for the diagnosis of a disease known as a disease (eg, cancer) involving the specific behavior of KRS. In addition, since it is specifically targeted to the KRS N-terminal region in vivo, it can be used as a therapeutic agent because it has an excellent effect of inhibiting cancer metastasis by inhibiting the interaction of the laminin receptor with the KRS N-terminal region, so it can be used as a therapeutic agent. Big.

<110> Medicinal Bioconvergence Research Center <120> Antibody specifically binding to the N-terminal region of lysyl-          tRNA synthetase exposed at cell outer membrane <130> NP17-0004P2 <150> KR 10-2017-0038775 <151> 2017-03-27 <150> KR 10-2017-0118890 <151> 2017-09-15 <160> 114 <170> KoPatentIn 3.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> N3 VH CDR1 <400> 1 Ser Tyr Asp Met Ser   1 5 <210> 2 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> N3 VH CDR1 <400> 2 agttatgata tgagc 15 <210> 3 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> N3 VH CDR2 <400> 3 Ala Ile Ser Tyr Asp Asn Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly     <210> 4 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> N3 VH CDR2 <400> 4 gcgatctctt atgataatgg taatacatat tacgctgatt ctgtaaaagg t 51 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> N3 VH CDR3 <400> 5 Met Ala Leu Asp Phe Asp Tyr   1 5 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> N3 VH CDR3 <400> 6 atggcgcttg atttcgacta c 21 <210> 7 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> N3 VL CDR1 <400> 7 Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Thr   1 5 10 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> N3 VL CDR1 <400> 8 tcttcatcta atattggcag taattatgtc acc 33 <210> 9 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> N3 VL CDR2 <400> 9 Asp Asn Ser Asn Arg Pro Ser   1 5 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> N3 VL CDR2 <400> 10 gataatagta atcggccaag c 21 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> N3 VL CDR3 <400> 11 Ala Ser Trp Asp Asp Ser Leu Ser Ala   1 5 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> N3 VL CDR3 <400> 12 gcttcttggg atgatagcct gagtgct 27 <210> 13 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> N5 VH CDR1 <400> 13 Asp Tyr Ala Met Ser   1 5 <210> 14 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> N5 VH CDR1 <400> 14 gattatgcta tgagc 15 <210> 15 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> N5 VH CDR2 <400> 15 Trp Ile Tyr Ser Gly Ser Gly Asn Lys Tyr Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly     <210> 16 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> N5 VH CDR2 <400> 16 tggatctatt ctggtagtgg taataaatat tacgctgatt ctgtaaaagg t 51 <210> 17 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> N5 VH CDR3 <400> 17 Met Gly Leu Asp Phe Asp Tyr   1 5 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> N5 VH CDR3 <400> 18 atgggtttgg atttcgacta c 21 <210> 19 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> N5 VL CDR1 <400> 19 Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Ser   1 5 10 <210> 20 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> N5 VL CDR1 <400> 20 tcttcatcta atattggcaa taattatgtc tcc 33 <210> 21 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> N5 VL CDR2 <400> 21 Asp Asp Asn Gln Arg Pro Ser   1 5 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> N5 VL CDR2 <400> 22 gatgataatc agcggccaag c 21 <210> 23 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> N5 VL CDR3 <400> 23 Gly Thr Trp Asp Asp Ser Leu Ser Ala   1 5 <210> 24 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> N5 VL CDR3 <400> 24 ggtacttggg atgatagcct gagtgct 27 <210> 25 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> N7 VH CDR1 <400> 25 Asp Tyr Tyr Met Ser   1 5 <210> 26 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> N7 VH CDR1 <400> 26 gattattata tgagc 15 <210> 27 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> N7 VH CDR2 <400> 27 Gly Ile Tyr Ser Gly Thr Gly Ser Ile Tyr Tyr Ala Asp Ser Val Glu   1 5 10 15 Gly     <210> 28 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> N7 VH CDR2 <400> 28 gggatctatt ctggtactgg tagtatatat tacgctgatt ctgtagaagg t 51 <210> 29 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> N7 VH CDR3 <400> 29 Pro Pro Tyr His Phe Asp Tyr   1 5 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> N7 VH CDR3 <400> 30 cctccgtatc atttcgacta c 21 <210> 31 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> N7 VL CDR1 <400> 31 Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Thr   1 5 10 <210> 32 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> N7 VL CDR1 <400> 32 tcttcatcta atattggcag taattatgtc acc 33 <210> 33 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> N7 VL CDR2 <400> 33 Ala Asp Ser Asn Arg Pro Ser   1 5 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> N7 VL CDR2 <400> 34 gctgatagta atcggccaag c 21 <210> 35 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> N7 VL CDR3 <400> 35 Gly Ala Trp Asp Tyr Ser Leu Ser Gly   1 5 <210> 36 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> N7 VL CDR3 <400> 36 ggtgcttggg attatagcct gagtggt 27 <210> 37 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> N9 VH CDR1 <400> 37 Asn Tyr Asp Met Ser   1 5 <210> 38 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> N9 VH CDR1 <400> 38 aattatgata tgagc 15 <210> 39 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> N9 VH CDR2 <400> 39 Trp Ile Tyr Ser Gly Asp Ser Ser Lys Tyr Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly     <210> 40 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> N9 VH CDR2 <400> 40 tggatctatt ctggtgatag tagtaaatat tacgctgatt ctgtaaaagg t 51 <210> 41 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> N9 VH CDR3 <400> 41 Glu Thr Arg Thr Phe Asp Tyr   1 5 <210> 42 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> N9 VH CDR3 <400> 42 gagacgcgga cgttcgacta c 21 <210> 43 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> N9 VL CDR1 <400> 43 Ser Ser Phe Asn Ile Gly Ser Asn Ala Val Tyr   1 5 10 <210> 44 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> N9 VL CDR1 <400> 44 tcttcattta atattggcag taatgctgtc tac 33 <210> 45 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> N9 VL CDR2 <400> 45 Tyr Asn Ser Gln Arg Pro Ser   1 5 <210> 46 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> N9 VL CDR2 <400> 46 tataatagtc agcggccaag c 21 <210> 47 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> N9 VL CDR3 <400> 47 Gly Ser Trp Asp Ala Ser Leu Ser Gly   1 5 <210> 48 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> N9 VL CDR3 <400> 48 ggctcttggg atgctagcct gagtggt 27 <210> 49 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> N3 VH <400> 49 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr              20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Ala Ile Ser Tyr Asp Asn Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Ser                  85 90 95 Ala Arg Met Ala Leu Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser         115 <210> 50 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> N3 VH <400> 50 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agttatgata tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagcg atctcttatg ataatggtaa tacatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactctgc gagaatggcg 300 cttgatttcg actactgggg ccagggtaca ctggtcaccg tgagctca 348 <210> 51 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> N3 VL <400> 51 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln   1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn              20 25 30 Tyr Val Thr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Asp Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser      50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg  65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Asp Ser Leu                  85 90 95 Ser Ala Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 110 <210> 52 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> N3 VL <400> 52 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtactg gctcttcatc taatattggc agtaattatg tcacctggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat gataatagta atcggccaag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggctgatta ttactgtgct tcttgggatg atagcctgag tgcttatgtc 300 ttcggcggag gcaccaagct gacggtccta 330 <210> 53 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> N5 VH <400> 53 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr              20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Trp Ile Tyr Ser Gly Ser Gly Asn Lys Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Met Gly Leu Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser         115 <210> 54 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> N5 VH <400> 54 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc gattatgcta tgagctgggt ccgccaggct 120 ccagggaaag ggctggagtg ggtctcatgg atctattctg gtagtggtaa taaatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagaatgggt 300 ttggatttcg actactgggg ccagggtaca ctggtcaccg tgagctca 348 <210> 55 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> N5 VL <400> 55 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln   1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Asn Asn              20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Asp Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser      50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg  65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Asp Ser Leu                  85 90 95 Ser Ala Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 110 <210> 56 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> N5 VL <400> 56 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtactg gctcttcatc taatattggc aataattatg tctcctggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat gatgataatc agcggccaag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggctgatta ttactgtggt acttgggatg atagcctgag tgcttatgtc 300 ttcggcggag gcaccaagct gacggtccta 330 <210> 57 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> N7 VH <400> 57 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr              20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Gly Ile Tyr Ser Gly Thr Gly Ser Ile Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Ser                  85 90 95 Ala Arg Pro Pro Tyr His Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser         115 <210> 58 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> N7 VH <400> 58 gaggtgcagc tgttggagtc ggggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc gattattata tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcaggg atctattctg gtactggtag tatatattac 180 gctgattctg tagaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactctgc gagacctccg 300 tatcatttcg actactgggg ccagggtaca ctggtcaccg tgagctca 348 <210> 59 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> N7 VL <400> 59 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln   1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn              20 25 30 Tyr Val Thr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Ala Asp Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser      50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg  65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Trp Asp Tyr Ser Leu                  85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 110 <210> 60 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> N7 VL <400> 60 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtactg gctcttcatc taatattggc agtaattatg tcacctggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat gctgatagta atcggccaag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggacg aggctgatta ttactgtggt gcttgggatt atagcctgag tggttatgtc 300 ttcggcggag gcaccaagct gacggtccta 330 <210> 61 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> N9 VH <400> 61 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr              20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Trp Ile Tyr Ser Gly Asp Ser Ser Lys Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Ser                  85 90 95 Ala Arg Glu Thr Arg Thr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser         115 <210> 62 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> N9 VH <400> 62 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc aattatgata tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcatgg atctattctg gtgatagtag taaatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactctgc gagagagacg 300 cggacgttcg actactgggg ccagggtaca ctggtcaccg tgagctca 348 <210> 63 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> N9 VL <400> 63 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln   1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Phe Asn Ile Gly Ser Asn              20 25 30 Ala Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Tyr Asn Ser Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser      50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg  65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Ala Ser Leu                  85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 110 <210> 64 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> N9 VL <400> 64 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtactg gctcttcatt taatattggc agtaatgctg tctactggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat tataatagtc agcggccaag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggctgatta ttactgtggc tcttgggatg ctagcctgag tggttatgtc 300 ttcggcggag gcaccaagct gacggtccta 330 <210> 65 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> linker for scFv <400> 65 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser   1 5 10 15 <210> 66 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> linker for scFv <400> 66 ggtggaggcg gttcaggcgg aggtggatcc ggcggtggcg gatcg 45 <210> 67 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> N3 scFv (VH + linker + VL) <400> 67 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr              20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Ala Ile Ser Tyr Asp Asn Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Ser                  85 90 95 Ala Arg Met Ala Leu Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly         115 120 125 Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr     130 135 140 Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile 145 150 155 160 Gly Ser Asn Tyr Val Thr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro                 165 170 175 Lys Leu Leu Ile Tyr Asp Asn Ser Asn Arg Pro Ser Gly Val Pro Asp             180 185 190 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser         195 200 205 Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp     210 215 220 Asp Ser Leu Ser Ala Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val 225 230 235 240 Leu     <210> 68 <211> 723 <212> DNA <213> Artificial Sequence <220> <223> N3 scFv (VH + linker + VL) <400> 68 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agttatgata tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagcg atctcttatg ataatggtaa tacatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactctgc gagaatggcg 300 cttgatttcg actactgggg ccagggtaca ctggtcaccg tgagctcagg tggaggcggt 360 tcaggcggag gtggatccgg cggtggcgga tcgcagtctg tgctgactca gccaccctca 420 gcgtctggga cccccgggca gagggtcacc atctcttgta ctggctcttc atctaatatt 480 ggcagtaatt atgtcacctg gtaccagcag ctcccaggaa cggcccccaa actcctcatc 540 tatgataata gtaatcggcc aagcggggtc cctgaccgat tctctggctc caagtctggc 600 acctcagcct ccctggccat cagtgggctc cggtccgagg atgaggctga ttattactgt 660 gcttcttggg atgatagcct gagtgcttat gtcttcggcg gaggcaccaa gctgacggtc 720 cta 723 <210> 69 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> N5 scFv (VH + linker + VL) <400> 69 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr              20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Trp Ile Tyr Ser Gly Ser Gly Asn Lys Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Met Gly Leu Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly         115 120 125 Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr     130 135 140 Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile 145 150 155 160 Gly Asn Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro                 165 170 175 Lys Leu Leu Ile Tyr Asp Asp Asn Gln Arg Pro Ser Gly Val Pro Asp             180 185 190 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser         195 200 205 Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp     210 215 220 Asp Ser Leu Ser Ala Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val 225 230 235 240 Leu     <210> 70 <211> 723 <212> DNA <213> Artificial Sequence <220> <223> N5 scFv (VH + linker + VL) <400> 70 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc gattatgcta tgagctgggt ccgccaggct 120 ccagggaaag ggctggagtg ggtctcatgg atctattctg gtagtggtaa taaatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagaatgggt 300 ttggatttcg actactgggg ccagggtaca ctggtcaccg tgagctcagg tggaggcggt 360 tcaggcggag gtggatccgg cggtggcgga tcgcagtctg tgctgactca gccaccctca 420 gcgtctggga cccccgggca gagggtcacc atctcttgta ctggctcttc atctaatatt 480 ggcaataatt atgtctcctg gtaccagcag ctcccaggaa cggcccccaa actcctcatc 540 tatgatgata atcagcggcc aagcggggtc cctgaccgat tctctggctc caagtctggc 600 acctcagcct ccctggccat cagtgggctc cggtccgagg atgaggctga ttattactgt 660 ggtacttggg atgatagcct gagtgcttat gtcttcggcg gaggcaccaa gctgacggtc 720 cta 723 <210> 71 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> N7 scFv (VH + linker + VL) <400> 71 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr              20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Gly Ile Tyr Ser Gly Thr Gly Ser Ile Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Ser                  85 90 95 Ala Arg Pro Pro Tyr His Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly         115 120 125 Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr     130 135 140 Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile 145 150 155 160 Gly Ser Asn Tyr Val Thr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro                 165 170 175 Lys Leu Leu Ile Tyr Ala Asp Ser Asn Arg Pro Ser Gly Val Pro Asp             180 185 190 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser         195 200 205 Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Trp Asp     210 215 220 Tyr Ser Leu Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val 225 230 235 240 Leu     <210> 72 <211> 723 <212> DNA <213> Artificial Sequence <220> <223> N7 scFv (VH + linker + VL) <400> 72 gaggtgcagc tgttggagtc ggggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc gattattata tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcaggg atctattctg gtactggtag tatatattac 180 gctgattctg tagaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactctgc gagacctccg 300 tatcatttcg actactgggg ccagggtaca ctggtcaccg tgagctcagg tggaggcggt 360 tcaggcggag gtggatccgg cggtggcgga tcgcagtctg tgctgactca gccaccctca 420 gcgtctggga cccccgggca gagggtcacc atctcttgta ctggctcttc atctaatatt 480 ggcagtaatt atgtcacctg gtaccagcag ctcccaggaa cggcccccaa actcctcatc 540 tatgctgata gtaatcggcc aagcggggtc cctgaccgat tctctggctc caagtctggc 600 acctcagcct ccctggccat cagtgggctc cggtccgagg acgaggctga ttattactgt 660 ggtgcttggg attatagcct gagtggttat gtcttcggcg gaggcaccaa gctgacggtc 720 cta 723 <210> 73 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> N9 scFv (VH + linker + VL) <400> 73 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr              20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Trp Ile Tyr Ser Gly Asp Ser Ser Lys Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Ser                  85 90 95 Ala Arg Glu Thr Arg Thr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly         115 120 125 Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr     130 135 140 Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Phe Asn Ile 145 150 155 160 Gly Ser Asn Ala Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro                 165 170 175 Lys Leu Leu Ile Tyr Tyr Asn Ser Gln Arg Pro Ser Gly Val Pro Asp             180 185 190 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser         195 200 205 Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp     210 215 220 Ala Ser Leu Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val 225 230 235 240 Leu     <210> 74 <211> 723 <212> DNA <213> Artificial Sequence <220> <223> N9 scFv (VH + linker + VL) <400> 74 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc aattatgata tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcatgg atctattctg gtgatagtag taaatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactctgc gagagagacg 300 cggacgttcg actactgggg ccagggtaca ctggtcaccg tgagctcagg tggaggcggt 360 tcaggcggag gtggatccgg cggtggcgga tcgcagtctg tgctgactca gccaccctca 420 gcgtctggga cccccgggca gagggtcacc atctcttgta ctggctcttc atttaatatt 480 ggcagtaatg ctgtctactg gtaccagcag ctcccaggaa cggcccccaa actcctcatc 540 tattataata gtcagcggcc aagcggggtc cctgaccgat tctctggctc caagtctggc 600 acctcagcct ccctggccat cagtgggctc cggtccgagg atgaggctga ttattactgt 660 ggctcttggg atgctagcct gagtggttat gtcttcggcg gaggcaccaa gctgacggtc 720 cta 723 <210> 75 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> membrane exposed Lysyl-tRNA synthetase N-term <400> 75 Met Ala Ala Val Gln Ala Ala Glu Val Lys Val Asp Gly Ser Glu Pro   1 5 10 15 Lys Leu Ser Lys Asn Glu Leu Lys Arg Arg Leu Lys Ala Glu Lys Lys              20 25 30 Val Ala Glu Lys Glu Ala Lys Gln Lys Glu Leu Ser Glu Lys Gln Leu          35 40 45 Ser Gln Ala Thr      50 <210> 76 <211> 597 <212> PRT <213> Artificial Sequence <220> <223> Lysyl-tRNA synthetase (KRS) full <400> 76 Met Ala Ala Val Gln Ala Ala Glu Val Lys Val Asp Gly Ser Glu Pro   1 5 10 15 Lys Leu Ser Lys Asn Glu Leu Lys Arg Arg Leu Lys Ala Glu Lys Lys              20 25 30 Val Ala Glu Lys Glu Ala Lys Gln Lys Glu Leu Ser Glu Lys Gln Leu          35 40 45 Ser Gln Ala Thr Ala Ala Ala Thr Asn His Thr Thr Asp Asn Gly Val      50 55 60 Gly Pro Glu Glu Glu Ser Val Asp Pro Asn Gln Tyr Tyr Lys Ile Arg  65 70 75 80 Ser Gln Ala Ile His Gln Leu Lys Val Asn Gly Glu Asp Pro Tyr Pro                  85 90 95 His Lys Phe His Val Asp Ile Ser Leu Thr Asp Phe Ile Gln Lys Tyr             100 105 110 Ser His Leu Gln Pro Gly Asp His Leu Thr Asp Ile Thr Leu Lys Val         115 120 125 Ala Gly Arg Ile His Ala Lys Arg Ala Ser Gly Gly Lys Leu Ile Phe     130 135 140 Tyr Asp Leu Arg Gly Glu Gly Val Lys Leu Gln Val Met Ala Asn Ser 145 150 155 160 Arg Asn Tyr Lys Ser Glu Glu Glu Phe Ile His Ile Asn Asn Lys Leu                 165 170 175 Arg Arg Gly Asp Ile Ile Gly Val Gln Gly Asn Pro Gly Lys Thr Lys             180 185 190 Lys Gly Glu Leu Ser Ile Ile Pro Tyr Glu Ile Thr Leu Leu Ser Pro         195 200 205 Cys Leu His Met Leu Pro His Leu His Phe Gly Leu Lys Asp Lys Glu     210 215 220 Thr Arg Tyr Arg Gln Arg Tyr Leu Asp Leu Ile Leu Asn Asp Phe Val 225 230 235 240 Arg Gln Lys Phe Ile Ile Arg Ser Lys Ile Ile Thr Tyr Ile Arg Ser                 245 250 255 Phe Leu Asp Glu Leu Gly Phe Leu Glu Ile Glu Thr Pro Met Met Asn             260 265 270 Ile Ile Pro Gly Gly Ala Val Ala Lys Pro Phe Ile Thr Tyr His Asn         275 280 285 Glu Leu Asp Met Asn Leu Tyr Met Arg Ile Ala Pro Glu Leu Tyr His     290 295 300 Lys Met Leu Val Val Gly Gly Ile Asp Arg Val Tyr Glu Ile Gly Arg 305 310 315 320 Gln Phe Arg Asn Glu Gly Ile Asp Leu Thr His Asn Pro Glu Phe Thr                 325 330 335 Thr Cys Glu Phe Tyr Met Ala Tyr Ala Asp Tyr His Asp Leu Met Glu             340 345 350 Ile Thr Glu Lys Met Val Ser Gly Met Val Lys His Ile Thr Gly Ser         355 360 365 Tyr Lys Val Thr Tyr His Pro Asp Gly Pro Glu Gly Gln Ala Tyr Asp     370 375 380 Val Asp Phe Thr Pro Pro Phe Arg Arg Ile Asn Met Val Glu Glu Leu 385 390 395 400 Glu Lys Ala Leu Gly Met Lys Leu Pro Glu Thr Asn Leu Phe Glu Thr                 405 410 415 Glu Glu Thr Arg Lys Ile Leu Asp Asp Ile Cys Val Ala Lys Ala Val             420 425 430 Glu Cys Pro Pro Pro Arg Thr Thr Ala Arg Leu Leu Asp Lys Leu Val         435 440 445 Gly Glu Phe Leu Glu Val Thr Cys Ile Asn Pro Thr Phe Ile Cys Asp     450 455 460 His Pro Gln Ile Met Ser Pro Leu Ala Lys Trp His Arg Ser Lys Glu 465 470 475 480 Gly Leu Thr Glu Arg Phe Glu Leu Phe Val Met Lys Lys Glu Ile Cys                 485 490 495 Asn Ala Tyr Thr Glu Leu Asn Asp Pro Met Arg Gln Arg Gln Leu Phe             500 505 510 Glu Glu Gln Ala Lys Ala Lys Ala Ala Gly Asp Asp Glu Ala Met Phe         515 520 525 Ile Asp Glu Asn Phe Cys Thr Ala Leu Glu Tyr Gly Leu Pro Pro Thr     530 535 540 Ala Gly Trp Gly Met Gly Ile Asp Arg Val Ala Met Phe Leu Thr Asp 545 550 555 560 Ser Asn Asn Ile Lys Glu Val Leu Leu Phe Pro Ala Met Lys Pro Glu                 565 570 575 Asp Lys Lys Glu Asn Val Ala Thr Thr Asp Thr Leu Glu Ser Thr Thr             580 585 590 Val Gly Thr Ser Val         595 <210> 77 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> N3 IgG heavy chain <400> 77 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr              20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Ala Ile Ser Tyr Asp Asn Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Ser                  85 90 95 Ala Arg Met Ala Leu Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala         115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu     130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser                 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu             180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr         195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr     210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro                 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val             260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr         275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val     290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser                 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro             340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val         355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly     370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp                 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His             420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys         435 440 445 <210> 78 <211> 1338 <212> DNA <213> Artificial Sequence <220> <223> N3 IgG heavy chain <400> 78 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agttatgata tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagcg atctcttatg ataatggtaa tacatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactctgc gagaatggcg 300 cttgatttcg actactgggg ccagggtaca ctggtcaccg tgagctcagc ctccaccaag 360 ggcccatcgg tcttccccct ggcaccctcc tccaagagca cctctggggg cacagcggcc 420 ctgggctgcc tggtcaagga ctacttcccc gaaccggtga cggtgtcgtg gaactcaggc 480 gccctgacca gcggcgtgca caccttcccg gctgtcctac agtcctcagg actctactcc 540 ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 600 gtgaatcaca agcccagcaa caccaaggtg gacaagaaag ttgagcccaa atcttgtgac 660 aaaactcaca catgcccacc gtgcccagca cctgaactcc tggggggacc gtcagtcttc 720 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 780 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 840 gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt 900 gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc 960 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg 1020 cagccccgag aaccacaggt gtataccctg cccccatccc gggatgagct gaccaagaac 1080 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1140 gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1200 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 1260 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 1320 tccctgtccc cgggtaaa 1338 <210> 79 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> N3 IgG light chain <400> 79 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln   1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn              20 25 30 Tyr Val Thr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Asp Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser      50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg  65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Asp Ser Leu                  85 90 95 Ser Ala Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg Thr             100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu         115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro     130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr                 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His             180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val         195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys     210 215 <210> 80 <211> 651 <212> DNA <213> Artificial Sequence <220> <223> N3 IgG light chain <400> 80 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtactg gctcttcatc taatattggc agtaattatg tcacctggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat gataatagta atcggccaag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggctgatta ttactgtgct tcttgggatg atagcctgag tgcttatgtc 300 ttcggcggag gcaccaagct gacggtccta cgtacggtgg ctgcaccatc tgtcttcatc 360 ttcccgccat ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat 420 aacttctatc ccagagaggc caaagtacag tggaaggtgg ataacgccct ccaatcgggt 480 aactcccagg agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc 540 accctgacgc tgagcaaagc agactacgag aaacacaaag tctacgcctg cgaagtcacc 600 catcagggcc tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg t 651 <210> 81 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> N5 IgG heavy chain <400> 81 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr              20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Trp Ile Tyr Ser Gly Ser Gly Asn Lys Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Met Gly Leu Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala         115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu     130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser                 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu             180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr         195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr     210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro                 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val             260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr         275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val     290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser                 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro             340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val         355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly     370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp                 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His             420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys         435 440 445 <210> 82 <211> 1338 <212> DNA <213> Artificial Sequence <220> <223> N5 IgG heavy chain <400> 82 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc gattatgcta tgagctgggt ccgccaggct 120 ccagggaaag ggctggagtg ggtctcatgg atctattctg gtagtggtaa taaatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagaatgggt 300 ttggatttcg actactgggg ccagggtaca ctggtcaccg tgagctcagc ctccaccaag 360 ggcccatcgg tcttccccct ggcaccctcc tccaagagca cctctggggg cacagcggcc 420 ctgggctgcc tggtcaagga ctacttcccc gaaccggtga cggtgtcgtg gaactcaggc 480 gccctgacca gcggcgtgca caccttcccg gctgtcctac agtcctcagg actctactcc 540 ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 600 gtgaatcaca agcccagcaa caccaaggtg gacaagaaag ttgagcccaa atcttgtgac 660 aaaactcaca catgcccacc gtgcccagca cctgaactcc tggggggacc gtcagtcttc 720 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 780 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 840 gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt 900 gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc 960 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg 1020 cagccccgag aaccacaggt gtataccctg cccccatccc gggatgagct gaccaagaac 1080 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1140 gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1200 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 1260 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 1320 tccctgtccc cgggtaaa 1338 <210> 83 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> N5 IgG light chain <400> 83 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln   1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Asn Asn              20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Asp Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser      50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg  65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Asp Ser Leu                  85 90 95 Ser Ala Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg Thr             100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu         115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro     130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr                 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His             180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val         195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys     210 215 <210> 84 <211> 651 <212> DNA <213> Artificial Sequence <220> <223> N5 IgG light chain <400> 84 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtactg gctcttcatc taatattggc aataattatg tctcctggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat gatgataatc agcggccaag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggctgatta ttactgtggt acttgggatg atagcctgag tgcttatgtc 300 ttcggcggag gcaccaagct gacggtccta cgtacggtgg ctgcaccatc tgtcttcatc 360 ttcccgccat ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat 420 aacttctatc ccagagaggc caaagtacag tggaaggtgg ataacgccct ccaatcgggt 480 aactcccagg agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc 540 accctgacgc tgagcaaagc agactacgag aaacacaaag tctacgcctg cgaagtcacc 600 catcagggcc tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg t 651 <210> 85 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> N7 IgG heavy chain <400> 85 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr              20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Gly Ile Tyr Ser Gly Thr Gly Ser Ile Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Ser                  85 90 95 Ala Arg Pro Pro Tyr His Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala         115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu     130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser                 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu             180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr         195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr     210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro                 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val             260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr         275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val     290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser                 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro             340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val         355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly     370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp                 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His             420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys         435 440 445 <210> 86 <211> 1338 <212> DNA <213> Artificial Sequence <220> <223> N7 IgG heavy chain <400> 86 gaggtgcagc tgttggagtc ggggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc gattattata tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcaggg atctattctg gtactggtag tatatattac 180 gctgattctg tagaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactctgc gagacctccg 300 tatcatttcg actactgggg ccagggtaca ctggtcaccg tgagctcagc ctccaccaag 360 ggcccatcgg tcttccccct ggcaccctcc tccaagagca cctctggggg cacagcggcc 420 ctgggctgcc tggtcaagga ctacttcccc gaaccggtga cggtgtcgtg gaactcaggc 480 gccctgacca gcggcgtgca caccttcccg gctgtcctac agtcctcagg actctactcc 540 ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 600 gtgaatcaca agcccagcaa caccaaggtg gacaagaaag ttgagcccaa atcttgtgac 660 aaaactcaca catgcccacc gtgcccagca cctgaactcc tggggggacc gtcagtcttc 720 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 780 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 840 gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt 900 gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc 960 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg 1020 cagccccgag aaccacaggt gtataccctg cccccatccc gggatgagct gaccaagaac 1080 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1140 gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1200 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 1260 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 1320 tccctgtccc cgggtaaa 1338 <210> 87 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> N7 IgG light chain <400> 87 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln   1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Asn Asn              20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Asp Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser      50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg  65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Asp Ser Leu                  85 90 95 Ser Ala Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg Thr             100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu         115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro     130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr                 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His             180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val         195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys     210 215 <210> 88 <211> 651 <212> DNA <213> Artificial Sequence <220> <223> N7 IgG light chain <400> 88 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtactg gctcttcatc taatattggc agtaattatg tcacctggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat gctgatagta atcggccaag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggacg aggctgatta ttactgtggt gcttgggatt atagcctgag tggttatgtc 300 ttcggcggag gcaccaagct gacggtccta cgtacggtgg ctgcaccatc tgtcttcatc 360 ttcccgccat ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat 420 aacttctatc ccagagaggc caaagtacag tggaaggtgg ataacgccct ccaatcgggt 480 aactcccagg agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc 540 accctgacgc tgagcaaagc agactacgag aaacacaaag tctacgcctg cgaagtcacc 600 catcagggcc tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg t 651 <210> 89 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> N9 IgG heavy chain <400> 89 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr              20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Trp Ile Tyr Ser Gly Asp Ser Ser Lys Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Ser                  85 90 95 Ala Arg Glu Thr Arg Thr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala         115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu     130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser                 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu             180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr         195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr     210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro                 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val             260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr         275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val     290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser                 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro             340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val         355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly     370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp                 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His             420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys         435 440 445 <210> 90 <211> 1338 <212> DNA <213> Artificial Sequence <220> <223> N9 IgG heavy chain <400> 90 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc aattatgata tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcatgg atctattctg gtgatagtag taaatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactctgc gagagagacg 300 cggacgttcg actactgggg ccagggtaca ctggtcaccg tgagctcagc ctccaccaag 360 ggcccatcgg tcttccccct ggcaccctcc tccaagagca cctctggggg cacagcggcc 420 ctgggctgcc tggtcaagga ctacttcccc gaaccggtga cggtgtcgtg gaactcaggc 480 gccctgacca gcggcgtgca caccttcccg gctgtcctac agtcctcagg actctactcc 540 ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 600 gtgaatcaca agcccagcaa caccaaggtg gacaagaaag ttgagcccaa atcttgtgac 660 aaaactcaca catgcccacc gtgcccagca cctgaactcc tggggggacc gtcagtcttc 720 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 780 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 840 gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt 900 gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc 960 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg 1020 cagccccgag aaccacaggt gtataccctg cccccatccc gggatgagct gaccaagaac 1080 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1140 gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1200 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 1260 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 1320 tccctgtccc cgggtaaa 1338 <210> 91 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> N9 IgG light chain <400> 91 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln   1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Phe Asn Ile Gly Ser Asn              20 25 30 Ala Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Tyr Asn Ser Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser      50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg  65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Ala Ser Leu                  85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg Thr             100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu         115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro     130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr                 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His             180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val         195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys     210 215 <210> 92 <211> 651 <212> DNA <213> Artificial Sequence <220> <223> N9 IgG light chain <400> 92 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtactg gctcttcatt taatattggc agtaatgctg tctactggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat tataatagtc agcggccaag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggctgatta ttactgtggc tcttgggatg ctagcctgag tggttatgtc 300 ttcggcggag gcaccaagct gacggtccta cgtacggtgg ctgcaccatc tgtcttcatc 360 ttcccgccat ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat 420 aacttctatc ccagagaggc caaagtacag tggaaggtgg ataacgccct ccaatcgggt 480 aactcccagg agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc 540 accctgacgc tgagcaaagc agactacgag aaacacaaag tctacgcctg cgaagtcacc 600 catcagggcc tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg t 651 <210> 93 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> scFv VH primer Forward <400> 93 agagagtgta cactcccagg cggccgaggt gcag 34 <210> 94 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> scFv VH primer Reverse <400> 94 cgccgctggg cccttggtgg aggctgagct cacggtgacc ag 42 <210> 95 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> scFv VL primer Forward <400> 95 aagcggccgc caccatggga tggagctgta tcatcctctt cttggtagca acagctacag 60 gtgtacactc ccagtctgtg ctgactcag 89 <210> 96 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> scFv VL primer Reverse <400> 96 cgccgccgta cgtaggaccg tcagcttggt 30 <210> 97 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> main binding site of N3 Ab <400> 97 Lys Leu Ser Lys Asn Glu Leu Lys Arg Arg Leu Lys Ala   1 5 10 <210> 98 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> N3 epitope F1 <400> 98 Met Ala Ala Val Gln Ala Ala Glu Val Lys Val Asp Gly Ser Glu Pro   1 5 10 15 Lys Leu Ser Lys Asn Glu Leu Lys Arg Arg Leu Lys Ala              20 25 <210> 99 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> N3 epitope F2 <400> 99 Gln Ala Ala Glu Val Lys Val Asp Gly Ser Glu Pro Lys Leu Ser Lys   1 5 10 15 Asn Glu Leu Lys Arg Arg Leu Lys Ala Glu Lys Lys Val Ala              20 25 30 <210> 100 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> N3 epitope F3 <400> 100 Lys Val Asp Gly Ser Glu Pro Lys Leu Ser Lys Asn Glu Leu Lys Arg   1 5 10 15 Arg Leu Lys Ala Glu Lys Lys Val Ala Glu Lys Glu Ala              20 25 <210> 101 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> N3 epitope F4 <400> 101 Glu Pro Lys Leu Ser Lys Asn Glu Leu Lys Arg Arg Leu Lys Ala Glu   1 5 10 15 Lys Lys Val Ala Glu Lys Glu Ala Lys Gln Lys Glu              20 25 <210> 102 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> N3 epitope F5 <400> 102 Lys Arg Arg Leu Lys Ala Glu Lys Lys Val Ala Glu Lys Glu Ala Lys   1 5 10 15 Gln Lys Glu Leu Ser Glu Lys Gln Leu Ser              20 25 <210> 103 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> mouse N3 epitope F1 (mF1) <400> 103 Met Ala Thr Leu Gln Glu Ser Glu Val Lys Val Asp Gly Glu Gln Lys   1 5 10 15 Leu Ser Lys Asn Glu Leu Lys Arg Arg Leu Lys Ala              20 25 <210> 104 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> mouse N3 epitope F2 (mF2) <400> 104 Gln Glu Ser Glu Val Lys Val Asp Gly Glu Gln Lys Leu Ser Lys Asn   1 5 10 15 Glu Leu Lys Arg Arg Leu Lys Ala Glu Lys Lys Leu Ala              20 25 <210> 105 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> mouse N3 epitope F3 (mF3) <400> 105 Lys Val Asp Gly Glu Gln Lys Leu Ser Lys Asn Glu Leu Lys Arg Arg   1 5 10 15 Leu Lys Ala Glu Lys Lys Leu Ala Glu Lys Glu Ala              20 25 <210> 106 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> mouse N3 epitope F4 (mF4) <400> 106 Gln Lys Leu Ser Lys Asn Glu Leu Lys Arg Arg Leu Lys Ala Glu Lys   1 5 10 15 Lys Leu Ala Glu Lys Glu Ala Lys Gln Lys Glu              20 25 <210> 107 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> mouse N3 epitope F5 (mF5) <400> 107 Arg Arg Leu Lys Ala Glu Lys Lys Leu Ala Glu Lys Glu Ala Lys Gln   1 5 10 15 Lys Glu Leu Ser Glu Lys Gln Leu Asn              20 25 <210> 108 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> rat N3 epitope F1 (rF1) <400> 108 Met Ala Thr Leu Arg Glu Gly Glu Val Lys Leu Asp Gly Glu Pro Lys   1 5 10 15 Leu Ser Lys Asn Glu Leu Lys Arg Arg Leu Lys Ala              20 25 <210> 109 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> rat N3 epitope F2 (rF2) <400> 109 Arg Glu Gly Glu Val Lys Leu Asp Gly Glu Pro Lys Leu Ser Lys Asn   1 5 10 15 Glu Leu Lys Arg Arg Leu Lys Ala Glu Lys Lys Lys Leu Ala              20 25 <210> 110 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> rat N3 epitope F3 (rF3) <400> 110 Lys Leu Asp Gly Glu Pro Lys Leu Ser Lys Asn Glu Leu Lys Arg Arg   1 5 10 15 Leu Lys Ala Glu Lys Lys Leu Ala Glu Lys Glu Ala              20 25 <210> 111 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> rat N3 epitope F4 (rF4) <400> 111 Pro Lys Leu Ser Lys Asn Glu Leu Lys Arg Arg Leu Lys Ala Glu Lys   1 5 10 15 Lys Leu Ala Glu Lys Glu Ala Lys Gln Lys Glu              20 25 <210> 112 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> rat N3 epitope F5 (rF5) <400> 112 Arg Arg Leu Lys Ala Glu Lys Lys Leu Ala Glu Lys Glu Ala Lys Gln   1 5 10 15 Lys Glu Leu Ser Glu Lys Gln Leu Asn              20 25 <210> 113 <211> 48 <212> PRT <213> Artificial Sequence <220> <223> Lysyl-tRNA synthetase N-term (mouse) <400> 113 Met Ala Thr Leu Gln Glu Ser Glu Val Lys Val Asp Gly Glu Gln Lys   1 5 10 15 Leu Ser Lys Asn Glu Leu Lys Arg Arg Leu Lys Ala Glu Lys Lys Leu              20 25 30 Ala Glu Lys Glu Ala Lys Gln Lys Glu Leu Ser Glu Lys Gln Leu Asn          35 40 45 <210> 114 <211> 48 <212> PRT <213> Artificial Sequence <220> <223> Lysyl-tRNA synthetase N-term (rat) <400> 114 Met Ala Thr Leu Arg Glu Gly Glu Val Lys Leu Asp Gly Glu Pro Lys   1 5 10 15 Leu Ser Lys Asn Glu Leu Lys Arg Arg Leu Lys Ala Glu Lys Lys Leu              20 25 30 Ala Glu Lys Glu Ala Lys Gln Lys Glu Leu Ser Glu Lys Gln Leu Asn          35 40 45

Claims (16)

Lysyl-tRNA synthetase (KRS, Lysyl-tRNA synthetase) specifically binds to an epitope consisting of any one sequence selected from the group consisting of SEQ ID NO: 101, SEQ ID NO: 106 and SEQ ID NO: 111 from the N-terminal Antibodies or fragments thereof.
delete According to claim 1, wherein the antibody or fragment thereof is SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 25 and SEQ ID NO: 37 consisting of an amino acid sequence selected from the group consisting of heavy chain complementarity determining region 1 (CDR1); Heavy chain complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 15, SEQ ID NO: 27 and SEQ ID NO: 39; A heavy chain variable region (VH) comprising a heavy chain complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 17, SEQ ID NO: 29 and SEQ ID NO: 41, and
Light chain complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 19, SEQ ID NO: 31 and SEQ ID NO: 43; Light chain complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 21, SEQ ID NO: 33 and SEQ ID NO: 45; Light chain variable region (VL) comprising a light chain complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 23, SEQ ID NO: 35 and SEQ ID NO: 47
Antibodies or fragments thereof.
According to claim 3, wherein the antibody or fragment thereof is a heavy chain complementarity determining region 1 comprising the amino acid sequence represented by SEQ ID NO: 1, heavy chain complementarity determining region 2 and SEQ ID NO: 5 comprising the amino acid sequence represented by SEQ ID NO: 3 Heavy chain variable region comprising heavy chain complementarity determining region 3 comprising the amino acid sequence displayed and light chain complementarity determining region comprising the amino acid sequence represented by SEQ ID NO: 7, light chain complementarity determination comprising the amino acid sequence represented by SEQ ID NO: 9 A light chain variable region comprising a light chain complementarity determining region 3 comprising an amino acid sequence represented by region 2 and SEQ ID NO: 11;
Heavy chain complementarity determining region 1 comprising the amino acid sequence represented by SEQ ID NO: 13, heavy chain complementarity determining region 2 comprising the amino acid sequence represented by SEQ ID NO: 15 and heavy chain complementarity determining region 3 comprising the amino acid sequence represented by SEQ ID NO: 17 Light chain complementarity determining region 1 comprising the heavy chain variable region and the amino acid sequence represented by SEQ ID NO: 19, light chain complementarity determining region 2 comprising the amino acid sequence represented by SEQ ID NO: 21 and the amino acid sequence represented by SEQ ID NO: 23 A light chain variable region comprising a light chain complementarity determining region 3 comprising;
Heavy chain complementarity determining region 1 comprising an amino acid sequence represented by SEQ ID NO: 25, heavy chain complementarity determining region 2 comprising an amino acid sequence represented by SEQ ID NO: 27 and heavy chain complementarity determining region 3 comprising an amino acid sequence represented by SEQ ID NO: 29 Light chain complementarity determining region 1 comprising the heavy chain variable region and the amino acid sequence represented by SEQ ID NO: 31, light chain complementarity determining region 2 comprising the amino acid sequence represented by SEQ ID NO: 33 and the amino acid sequence represented by SEQ ID NO: 35 A light chain variable region comprising a light chain complementarity determining region 3 comprising; And
Heavy chain complementarity determining region 1 comprising the amino acid sequence represented by SEQ ID NO: 37, heavy chain complementarity determining region 2 comprising the amino acid sequence represented by SEQ ID NO: 39 and heavy chain complementarity determining region 3 comprising the amino acid sequence represented by SEQ ID NO: 41 Light chain complementarity determining region 1 comprising the heavy chain variable region comprising and the amino acid sequence represented by SEQ ID NO: 43, light chain complementarity determining region 2 comprising the amino acid sequence represented by SEQ ID NO: 45 and the amino acid sequence represented by SEQ ID NO: 47 A light chain variable region comprising a light chain complementarity determining region 3 comprising;
Antibodies or fragments comprising a heavy chain variable region and a light chain variable region selected from the group consisting of.
The method of claim 3, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57 and SEQ ID NO: 61, and the light chain variable region comprises SEQ ID NO: 51, SEQ ID NO: 55, An antibody or fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 59 and SEQ ID NO: 63.
The antibody of claim 3, wherein the antibody is selected from the group consisting of IgG, IgA, IgM, IgE and IgD, and the fragment is Diabody, Fab, Fab ', F (ab) 2, F (ab') 2, Fv. And a scFv antibody or fragment thereof.
7. The antibody or fragment thereof according to claim 6, wherein the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 and SEQ ID NO: 73.
A polynucleotide encoding the antibody or fragment thereof according to any one of claims 1 to 3 to 7.
Recombinant expression vector comprising the polynucleotide of claim 8.
A cell transformed with the recombinant expression vector of claim 9.
(a) transforming a host cell with the recombinant expression vector of claim 9;
(b) culturing the transformed host cell to produce an antibody or fragment thereof; And
(c) an antibody that specifically binds to the N-terminus of Lysyl-tRNA synthetase (KRS) exposed to the outer membrane, comprising the step of obtaining an antibody or fragment produced in the host cell, or How to make the fragment.
A lysyl-tRNA synthetase (KRS) N-terminal exposed to the outer membrane, comprising contacting the antibody of claim 1 or a fragment thereof with a sample and detecting the antibody or fragment thereof. Specific detection method.
A pharmaceutical composition for inhibiting cancer metastasis comprising the antibody of claim 1 or a fragment thereof as an active ingredient.
The method of claim 13, wherein the cancer is breast cancer, colon cancer, lung cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, Anal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer , Selected from the group consisting of prostate cancer, chronic or acute leukemia, lymphocyte lymphoma, bladder cancer, kidney or urinary tract cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma and pituitary adenoma Composition characterized in that.
14. The composition of claim 13, wherein the cancer is breast cancer or lung cancer.
A composition for diagnosing cancer comprising the antibody of claim 1 or a fragment thereof as an active ingredient.

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