KR20160093502A - anti-EPRS monoclonal antibody and uses thereof - Google Patents

Abstract

The present invention relates to an anti-EPRS antibody which specifically binds to EPRS (glycyl-tRNA synthetase). More specifically, the present invention relates to an antibody specifically binding to human EPRS or a fragment thereof, a production method thereof, and a pharmaceutical composition for diagnosing and treating cancer, inflammatory disease, or immunological disease, including the same. The antibody or the fragment thereof can specifically bind to the human EPRS, and does not have cross-reactivity with other proteins including the same ARS family, thereby detecting and inhibiting EPRS. Thus, the antibody or the fragment thereof can be used for detecting EPRS and also for diagnosing and treating cancer, inflammatory disease, or immunological disease associated with EPRS.

Description

Anti-EPRS monoclonal antibodies and uses thereof < RTI ID = 0.0 >

The present invention relates to an anti-EPRS antibody selectively binding to EPRS (glutamyl-prolyl tRNA synthetase), and more particularly to an antibody or fragment thereof that binds to human EPRS, a method for producing the antibody, and a pharmaceutical composition for treating cancer comprising the same .

Aminoacyl-tRNA synthetase (ARS) is an enzyme that attaches a specific amino acid to its corresponding tRNA. In the case of higher organisms, it consists of 23 enzymes including three enzymes involved in the formation of multisynthetase complexes such as AIMP1 (p43), (AIMP2) p38, and (AIMP3) Some enzymes exist in free form. However, in recent years, it has been reported that in addition to its basic functions, it has various other active functions in a specific environment, such as glutamyl-prolyl tRNA synthetase (EPRS). EPRS is a form in which two enzyme active domains are linked by a linker, and this linker has three domains, WHEP, repeated. The structure of this WHEP domain has a similar structure to some ARSs enzymes such as WRS, HRS, GRS, and MRS but does not have a repeating structure unlike EPRS. The relationship between EPRS and disease is due to interferon gamma (IFN-γ) binding to the 3-UTR portion of mRNA of various regulatory proteins to form gamma-interferon-activated inhibitor of translation complex (GAIT) (Sampath, P. et al.) Noncanonical function of glutamylprolyltRNA synthetase: gene-specific silencing of translation. Cell 119, 195, 208 (2004) ). In addition, the protein synthesis process of VEGF (Vascular Endothelial Growth Factor A), an important factor in angiogenesis, has also been shown to be regulated by EPRS (Ray, PS et al. 457, 915-919 (2009)). Therefore, EPRS may be involved as an important regulator of gene expression in cancer, immune and inflammatory diseases, and may be used as an important diagnostic biomarker.

However, despite its importance as a biomarker for ARS, including EPRS, ARSs have similarity in protein structure, so that the antibody obtained from an animal's immune response cross-reacts with other ARSs, and no high-sensitivity antibody is produced many. The antibodies of the present invention are expected to be highly diagnostic and industrially applicable antibodies as well as for research because they lack excellent sensitivity and cross-reactivity between ARS.

The present inventors investigated antibodies specifically binding to EPRS, and found fragments specifically binding to EPRS in a library made by the phage display method, and their sequences and binding specificities were found, thereby completing the present invention.

It is therefore an object of the present invention to provide antibodies or fragments thereof that bind to human EPRS.

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

Another object of the present invention is to provide a vector comprising the polynucleotide.

Another object of the present invention is to provide a cell comprising said vector.

It is another object of the present invention to provide a method for producing an antibody or a fragment thereof that binds to human EPRS.

Another object of the present invention is to provide an EPRS-specific detection method comprising contacting an antibody or a fragment thereof with a sample and detecting the antibody or fragment thereof.

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

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

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating immunological diseases comprising the antibody or fragment thereof as an active ingredient.

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

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating an inflammatory disease comprising the antibody or the fragment thereof as an active ingredient.

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

(CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 1, a complementarity determining region (CDR) L2 including the amino acid sequence represented by SEQ ID NO: 2, An antibody light chain variable region (VL) comprising a complementary crystal region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 3 and a complementary crystal region (CDR) H1 comprising an amino acid sequence represented by SEQ ID NO: An antibody heavy chain variable region (VH) comprising a complementary crystal region (CDR) H2 comprising an amino acid sequence represented by SEQ ID NO: 5 and a complementary crystal region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 6;

ii) a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 15, a complementarity determining region (CDR) L2 including the amino acid sequence represented by SEQ ID NO: 16, and an amino acid sequence represented by SEQ ID NO: An antibody light chain variable region (VL) comprising the complementary crystal region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 18, a complementary crystal region (CDR) H1 comprising the amino acid sequence represented by SEQ ID NO: 18, An antibody heavy chain variable region (VH) comprising a crystal region (CDR) H2, a complementary crystal region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 20; And

iii) a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 29, a complementarity determining region (CDR) L2 including the amino acid sequence represented by SEQ ID NO: 30, and an amino acid sequence represented by SEQ ID NO: 31 An antibody light chain variable region (VL) comprising the complementary crystal region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 32 and a complementary crystal region (CDR) H1 comprising the amino acid sequence represented by SEQ ID NO: An antibody heavy chain variable region (VH) comprising a crystal region (CDR) H2, a complementary crystal region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 34;

Lt; RTI ID = 0.0 > EPRS < / RTI >

In order to achieve another object of the present invention, the present invention provides a polynucleotide encoding said antibody or fragment thereof.

In order to achieve another object of the present invention, the present invention provides a vector comprising the polynucleotide.

In order to achieve another object of the present invention, the present invention provides a cell comprising the vector.

In order to accomplish another object of the present invention, the present invention provides a method for producing a polypeptide comprising the steps of: culturing the above cells under a condition that a polynucleotide is expressed to produce a polypeptide comprising a light chain and a heavy chain variable region; And recovering the polypeptide. The present invention also provides a method for producing an antibody binding to human EPRS or a fragment thereof.

In order to achieve another object of the present invention, there is provided an EPRS-specific detection method comprising contacting an antibody or a fragment thereof with a sample and detecting the antibody or fragment thereof.

In order to accomplish another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer 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 fragment thereof as an active ingredient.

In order to accomplish another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating immunological diseases comprising the antibody or fragment thereof as an active ingredient.

In order to accomplish another object of the present invention, there is provided a composition for diagnosing an immune disease comprising the antibody or the fragment thereof as an active ingredient.

In order to accomplish another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease 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 an inflammatory disease comprising the antibody or fragment thereof as an active ingredient.

Hereinafter, the present invention will be described in detail.

(CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 1, a complementarity determining region (CDR) L2 including the amino acid sequence represented by SEQ ID NO: 2, an amino acid represented by SEQ ID NO: An antibody light chain variable region (VL) comprising a complementary crystal region (CDR) L3 comprising a sequence and a complementary crystal region (CDR) H1 comprising an amino acid sequence represented by SEQ ID NO: 4, an amino acid sequence represented by SEQ ID NO: 5 An antibody heavy chain variable region (VH) comprising a complementarity determining region (CDR) H2 comprising the amino acid sequence shown in SEQ ID NO: 6, and a complementary crystal region (CDR) H3 comprising the amino acid sequence represented by SEQ ID NO: 6;

ii) a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 15, a complementarity determining region (CDR) L2 including the amino acid sequence represented by SEQ ID NO: 16, and an amino acid sequence represented by SEQ ID NO: An antibody light chain variable region (VL) comprising the complementary crystal region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 18, a complementary crystal region (CDR) H1 comprising the amino acid sequence represented by SEQ ID NO: 18, An antibody heavy chain variable region (VH) comprising a crystal region (CDR) H2, a complementary crystal region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 20; And

iii) a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 29, a complementarity determining region (CDR) L2 including the amino acid sequence represented by SEQ ID NO: 30, and an amino acid sequence represented by SEQ ID NO: 31 An antibody light chain variable region (VL) comprising the complementary crystal region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 32 and a complementary crystal region (CDR) H1 comprising the amino acid sequence represented by SEQ ID NO: (CDR) H2, and an antibody heavy chain variable region (VH) comprising a complementary crystal region (CDR) H3 comprising the amino acid sequence of SEQ ID NO: 34. Antibody or fragment thereof.

Glutamyl-prolyl tRNA synthetase (EPRS) is an enzyme that catalyzes the binding of tRNAs of glutamyl and glycyl, and is a kind of ARS (Aminoacyl-tRNA synthetase). The EPRS of the invention generally refers to native or recombinant human EPRS, and non-human homologues of human EPRS.

The terms "antibody", "anti-EPRS antibody", "humanized anti-EPRS antibody" and "modified anti-EPRS antibody" and "anti-EPRS antibody" are used in the broadest sense in the present invention, (Such as monoclonal antibodies, full length monoclonal antibodies), polyclonal antibodies (polyclonal antibodies), multispecific antibodies (e. G., Bispecific antibodies), and antibody fragments (E. G., Binding to < / RTI > EPRS).

The antibody of the present invention is an antibody in which a specific amino acid sequence is contained in a light chain and a heavy chain CDR so as to be capable of selectively binding to EPRS, and includes both monoclonal antibodies and polyclonal antibodies, preferably monoclonal antibodies have. In addition, the antibody of the present invention includes both a chimeric antibody, a humanized antibody, and a human antibody, and may preferably be a human antibody.

A monoclonal antibody of the invention refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies bind to single antigen epitopes very specifically.

The term "monoclonal" refers to the character of an antibody, such as an antibody is obtained from a substantial homologous population, and does not necessarily mean that the antibody has to be produced by a particular method.

For example, the monoclonal antibodies of the present invention can be prepared as described in Kohler et al. (1975) Nature 256: 495, or may be prepared by recombinant DNA methods (see U.S. Patent No. 4,816,567). See also, for example, Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597 and Presta (2005) J. Allergy Clin. Immunol. 116: 731). ≪ / RTI >

The antibody of the present invention specifically includes a chimeric antibody, wherein a portion of the heavy chain and / or light chain originates from a particular species or is homologous or homologous to the corresponding sequence of a particular antibody, May be of another species or may be identical or homologous to the corresponding sequence of another antibody, as long as it exhibits the desired biological activity (e. G., Selective binding to EPRS) (see U.S. Patent No. 4,816,567; And Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855].

Humanized antibodies are antibodies that include both human and non-human (e.g., rat, rat) antibodies. Generally, the remainder of the epitope binding site (CDR) is of human antibodies, (CDR) may comprise a non-human derived sequence.

A complete human antibody refers to an antibody comprising only a human immunoglobulin protein sequence and can be produced in a hybridoma originating from a mouse, mouse cell, or mouse cell, or produced by a phage display method.

Natural antibodies produced in vivo are typically about 150,000 daltons, heterotetrameric glycoproteins, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by one covalent disulfide bond, but the disulfide chain number varies between the heavy chains of the different immunoglobulin isoforms. Each heavy and light chain also has regularly spaced intra-chain disulfide bridges. Each heavy chain has a variable domain (VH) followed by a number of constant domains at one end. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; The constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. It is believed that a particular amino acid residue forms an interface between the light chain variable domain and the heavy chain variable domain.

"Variable domain" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable region of the heavy chain is referred to as "VH" or "VH", and the variable region of the light chain is referred to as "VL" or "VL". These domains are generally the most variable part of the antibody and comprise an antigen binding site.

The term " hypervariable "means that some of the sequences in the variable region are broadly different in sequence between the antibodies and have residues that are directly related to the binding and specificity of each particular antibody to its specific antigenic determinants .

In both the light chain and heavy chain variable regions, the hypervariability is focused on three segments known as complementarity determining regions (CDRs) or hypervariable loops (HVLs). CDRs are described in Kabat et al., 1991, In: Sequences of Proteins of Immunological Interest, 5th Ed. HVL has been described by Chothia and Le et al., 1987, J. MoI., ≪ RTI ID = 0.0 > Biol. 196: 901-917, which is structurally defined according to the three-dimensional structure of the variable region. As defined by Kabat, CDR-L1 is located approximately at residues 24-34 in the light chain variable region, CDR-L2 is approximately at residues 50-56, CDRL3 is approximately at residues 89-97; CDR-H1 is located approximately at residues 31-35 in the heavy chain variable region, CDR-H2 at approximately residues 50-65, and CDR-H3 at approximately residues 95-102.

The three CDRs in each of the heavy and light chains are separated by a framework region (FR), which includes sequences that tend to be less variable. From the amino terminus to the carboxy terminus of the heavy and light chain variable regions, the FRs and CDRs are arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The large < RTI ID = 0.0 > ss < / RTI > sheet arrangement of FRs brings the CDRs within each chain closer to each other as well as to the CDRs from the other strands. The form produced contributes to the antigen binding site (see Kabat et al., 1991, NIH Publ. No. 91-3242, Vol. I, pages 647-669), but not all CDR residues need to be directly involved in antigen binding.

The antibody of the present invention is characterized in that each CDR belonging to the light chain and heavy chain variable regions each include a specific sequence and specifically binds to EPRS. Specifically, the antibody of the present invention comprises i) an amino acid sequence of SEQ ID NO: 1 A complementarity determining region (CDR) L1 comprising the sequence, a complementarity determining region (CDR) L2 including the amino acid sequence represented by SEQ ID NO: 2, and a complementarity determining region (CDR) L3 including the amino acid sequence represented by SEQ ID NO: (CDR) H1 comprising the amino acid sequence shown in SEQ ID NO: 4, a complementary crystal region (CDR) H2 comprising the amino acid sequence shown in SEQ ID NO: 5, an antibody light chain variable region An antibody heavy chain variable region (VH) comprising a complementary crystal region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 6;

ii) a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 15, a complementarity determining region (CDR) L2 including the amino acid sequence represented by SEQ ID NO: 16, and an amino acid sequence represented by SEQ ID NO: An antibody light chain variable region (VL) comprising the complementary crystal region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 18, a complementary crystal region (CDR) H1 comprising the amino acid sequence represented by SEQ ID NO: 18, An antibody heavy chain variable region (VH) comprising a crystal region (CDR) H2, a complementary crystal region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 20; And

iii) a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 29, a complementarity determining region (CDR) L2 including the amino acid sequence represented by SEQ ID NO: 30, and an amino acid sequence represented by SEQ ID NO: 31 An antibody light chain variable region (VL) comprising the complementary crystal region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 32 and a complementary crystal region (CDR) H1 comprising the amino acid sequence represented by SEQ ID NO: An antibody comprising an antibody heavy chain variable region (VH) comprising a crystal region (CDR) H2, and a complementary crystal region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 34.

The antibodies of the present invention may preferably comprise a specific light chain variable region and a heavy chain variable region,

i) an antibody comprising a 132 to 241 amino acid sequence of SEQ ID NO: 13 as a light chain variable region and a 1 to 116 amino acid sequence of SEQ ID NO: 13 as a heavy chain variable region;

ii) an antibody comprising a 132 to 241 amino acid sequence of SEQ ID NO: 27 as a light chain variable region and a 1 to 116 amino acid sequence of SEQ ID NO: 27 as a heavy chain variable region; And

iii) an antibody comprising the amino acid sequence from position 132 to position 241 of SEQ ID NO: 41 as the light chain variable region and the amino acid sequence from position 1 to 116 of SEQ ID NO: 41 as the heavy chain variable region;

≪ / RTI >

The antibody of the present invention may most preferably be an antibody comprising an amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 27 and SEQ ID NO: 41.

An antibody fragment, fragment or " fragment thereof " of the invention comprises at least a portion or variable region (e.g., one or more CDRs) of antigen binding, typically at least of the parent antibody, that retains at least a portion of the binding specificity of the parent antibody Fragments or derivatives of antibodies. Examples of antibody fragments include Fab, Fab ', F (ab') 2 and Fv fragments; Diabody; Linear antibodies; Single-chain antibody molecules, such as sc-Fv; And multispecific antibodies formed from antibody fragments. Typically, an antibody fragment or derivative retains at least 10% of its EPRS binding activity when the activity is expressed on a molar basis. Preferably, the antibody fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the EPRS binding affinity as the parent antibody. In addition, the EPRS antibody fragment may comprise conservative amino acid substitutions (referred to as conservative variants of the antibody) that do not substantially alter its biological activity. "Binding compounds" of the present invention refer to both antibodies and fragments thereof.

Fab consists of one light chain, and one heavy chain CH1 (first constant domain) and variable region. The heavy chain of a Fab molecule can not form a disulfide bond with another heavy chain molecule.

The Fc region contains two heavy chain fragments comprising the CH1 and CH2 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domain.

Fab 'contains one light chain, and a region between the VH and CH1 domains and the CH1 and CH2 domains, so that intrachain disulfide bonds are formed between the two heavy chains of the two Fab' fragments to form F (ab ') 2 molecules Lt; RTI ID = 0.0 > a < / RTI > heavy chain.

F (ab ') 2 contains two light chains, and two heavy chains containing a portion of the immobilized region between the CH1 and CH2 domains such that an intrachain disulfide bond is formed between the two heavy chains. Thus, the F (ab ') 2 fragment consists of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.

Fv is an antibody fragment that contains both heavy and light chain variable regions but lacks a fixed region.

A single-chain Fv or scFv represents an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains such that the scFv forms a preferred structure for antigen binding. For an overview of scFv, see Pluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315. See also International Patent Publication No. WO 88/01649 and U.S. Patent Nos. 4,946,778 and 5,260,203.

Diabody refers to small antibody fragments having two antigen-binding sites, wherein the fragment comprises a heavy chain variable domain (VH) (VH-VL) linked to a light chain variable domain (VL) in the same polypeptide chain. Using short linkers that do not allow pairing between two domains on the same chain, the domains are forcedly paired with the complementary domains of the other chain to generate two antigen-binding sites. Diabodies are described, for example, in EP 404,097; WO 93/11161; And Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

A linear antibody refers to an antibody comprising a pair of series Fd fragments (VH-CHl-VH-CHl) that form a pair of antigen binding sites. Linear antibodies are described, for example, in Zapata et al. 1995, Protein Eng. 8 (10): < RTI ID = 0.0 > 1057-1062. ≪ / RTI >

A "domain antibody" is an immunologically functional immunoglobulin fragment that contains only the variable region of the heavy chain or the variable region of the light chain.

In some instances, two or more VH regions are covalently linked to a peptide linker to produce a divalent domain antibody. The two VH regions of the divalent domain antibody can target the same or different antigens.

The bivalent antibody comprises two antigen binding sites. In some instances, the two binding regions have the same antigen specificity. However, the bivalent antibody may be bispecific.

The antibody or fragment thereof of the present invention can be produced by a method known in the art, for example, a phage display method or a yeast cell surface expression system. The scFv may be prepared by the methods described in U.S. Patent Nos. 4,946,778 and 5,258,498, and methods for recombinantly producing Fab, Fab 'and F (ab') 2 fragments include WO 92/22324 And the like can be used.

The antibody of the present invention may be derived from any animal including mammals including humans, birds, and the like. Preferably, the antibody may be an antibody of a human, a mouse, a donkey, a sheep, a rabbit, a goat, a guinea pig, a camel, a horse or a chicken.

A human antibody is an antibody having the amino acid sequence of a human immunoglobulin, including an antibody isolated from a human immunoglobulin library or an antibody isolated from an animal that is transgenic for one or more human immunoglobulin and does not express an endogenous immunoglobulin See Patent No. 5,939,598).

The antibody of the present invention may be conjugated with an enzyme, a fluorescent substance, a radioactive substance and a protein, but is not limited thereto. Methods of conjugating such materials to antibodies are also well known in the art.

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

Polynucleotides may be described as oligonucleotides or nucleic acids and may be described as DNA molecules (e.g., cDNA or genomic DNA, RNA molecules (e.g., mRNA), DNA generated using nucleotide analogs (E. G., Peptide nucleic acids and non-naturally occurring nucleotide analogs) and hybrids thereof. The polynucleotides may be single-stranded or double- stranded.

The polynucleotide of the present invention is not particularly limited as far as it encodes the antibody or fragment thereof of the present invention,

i) a polynucleotide represented by SEQ ID NO: 7 to 12;

ii) a polynucleotide represented by SEQ ID NOs: 21 to 26; And

iii) a polynucleotide represented by SEQ ID NO: 35 to 40; ≪ RTI ID = 0.0 > polynucleotides < / RTI >

Polynucleotides encoding the antibodies or fragments thereof of the present invention can be obtained by methods well known in the art. For example, an oligonucleotide synthesis technique well known in the art, for example, a polymerase chain reaction (PCR), or the like may be used based on a DNA sequence or a corresponding amino acid sequence encoding a part or all of the heavy chain and light chain of the antibody . ≪ / RTI >

The present invention also provides a vector comprising the polynucleotide.

The vector of the present invention is used for the purpose of cloning or expression of the polynucleotide of the present invention for recombinant production of the antibody or fragment thereof of the present invention and is generally composed of a signal sequence, a replication origin, one or more marker genes, an enhancer element, And a transcription termination sequence.

The vector of the present invention may preferably be an expression vector, and more preferably, may be a vector comprising a polynucleotide of the present invention operably linked to a regulatory sequence, for example, a promoter.

A plasmid, a type of vector, refers to a linear or circular double stranded DNA molecule to which external polynucleotide fragments can be ligated. Other forms of vectors are viral vectors (e. G., Replication defective retroviruses, adenoviruses and adenoassociated viruses), where additional DNA fragments Can be introduced into the viral genome. Certain vectors may be self-replicating within the host cells into which they are introduced (e.g., bacterial origin including bacterial origin and episomal mammalian vectors) (autonomous replication). Other vectors (e. G., Non-episomal mammalian vectors) are integrated into the genome of the host cell by introduction into the host cell, Are duplicated together.

An expression vector is a form of a vector capable of expressing a selected polynucleotide. One polynucleotide sequence is "operably linked" to the regulatory sequence when the regulatory sequence affects the expression (e.g., level, timing, or location of expression) of the polynucleotide sequence . The modulatory sequence is a sequence that affects the expression (e.g., level, timing, or location of expression) of the nucleic acid to which it is operatively linked. The modulation sequence can be, for example, a nucleic acid whose effect is directly or indirectly affected by the action of one or more other molecules (e. G., Polypeptides that bind to the regulatory sequence and / . The regulatory sequence includes promoters, enhancers, and other expression control elements. The vector of the present invention may preferably be a pCom3x (phagmid) vector containing scFv Insert at SfiI site.

The present invention also provides a cell comprising the vector of the present invention.

The cell of the present invention is not particularly limited so far as the type of cell surface that can be used to express the polynucleotide of the present invention.

The cells of the present invention can be produced by transforming a host cell with a prokaryotic (e. G., E. coli), a eukaryote (e. G. Yeast or other fungi), a plant cell (e. G., A tobacco or tomato plant cell) , A monkey cell, a hamster cell, a rat cell, a mouse cell or an insect cell) or a hybridoma.

Suitable prokaryotes for this purpose include gram-negative or gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, for example E. coli. For example, E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example Salmonella typhimurium, Serratia, For example, Serratia marcescans and Shigella, and Bacilli, for example, B. subtilis and rain. B. licheniformis, Pseudomonas, such as p. 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. Cola, but not limited to, for example, this. Coli ER2537, i. Coli B, Lee. E. coli X1776 (ATCC 31, 537); E. coli W3110 (ATCC 27,325) or LacZ can be expressed. Coli, and more preferably this. E. coli ER2537.

Saccharomyces cerevisiae is the most commonly used eukaryotic cell of the present invention. However, many other genera, species and strains, including, but not limited to, Schizosaccharomyces pombe, Kluyveromyces hosts, e.g., K. K.lactis, K. K. fragilis (ATCC 12, 424), Kay. K. bulgaricus (ATCC 16,045), Kay. K. wickeramii (ATCC 24,178), Kay. K. waltii (ATCC 56,500), Kay. K. drosophilarum (ATCC 36,906), Kay. K. thermotolerans and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Tricot Neurospora crassa; Schwanniomyces, such as Shibaniomycetes occidentalis; and filamentous fungi, such as Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, Such as Neurospora, Penicillium, Tolypocladium and Aspergillus hosts, such as, for example, A. nidulans and A. niger. It is possible.

The cells of the present invention may be animal cells, particularly vertebrate cells. In culture (tissue culture), the proliferation of vertebrate cells has become a routine method and techniques are widely available. Examples of useful mammalian host cells include, but are not limited to, lines of monkey kidney CV1 (COS-7, ATCC CRL 1651) transformed by SV40, 293 or 293 cells subcloned from human embryonic kidney lysates (BHK, ATCC CCL10), Chinese hamster ovary cells / -DHFR "(CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA murine Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23: 243-251), monkey kidney cells (CVl ATCC CCL 70), African green monkey kidney cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (ATCC CCL 75), human hepatocyte (Hep G2, HB 8065), mouse breast tumor (MMT 060562, ATCC CCL51), TRI cells (Mather et al., 1982, Annals NY. Acad. Sci. three A human embryonic kidney cell (HEK 293 cell), and an Exp293FTM cell, and may be a CHO cell, a HEK 293 cell (human embryonic kidney cell), or an Expi 293 FTM cell .

A cell of the present invention is a cultured cell that can be transformed or transfected with a polynucleotide of the present invention or a vector containing it, which can be subsequently expressed in the host cell. A recombinant cell refers to a cell transformed or transfected with a polynucleotide to be expressed. A cell of the invention may also be a cell that comprises a polynucleotide of the invention but does not express it at a desired level unless a regulatory sequence is introduced into the cell to operably link to the polynucleotide.

The cells of the present invention can be cultured in various media. (Sigma-Aldrich Co., St. Louis, MO), minimal essential medium (MEM, Sigma-Aldrich Co.), RPMI-1640 (Sigma-Aldrich Co.) And Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich Co.) are suitable for culturing cells. The medium may be supplemented with hormones and / or other growth factors, salts, buffers, nucleotides, antibiotics, trace elements and glucose or equivalent energy sources, if necessary.

The medium of the present invention may preferably be SB (Bactotrytone 30 g, yeast extract 20 g, MOPS buffer 10 g / L) Medium, FreeStyle TM 293 Medium or Expi 293 TM Medium.

In the meantime, the present invention includes a step of culturing the above cells under the condition that a polynucleotide is expressed, producing a polypeptide including a light chain and a heavy chain variable region, and recovering the polypeptide from the cell or a culture medium in which the polypeptide is cultured Lt; RTI ID = 0.0 > EPRS. ≪ / RTI >

The cells of the production method of the present invention are as described above and include a polynucleotide encoding the antibody of the present invention.

The polypeptide of the production method of the present invention may be an antibody of the present invention or a fragment thereof itself, and may further be an antibody or an amino acid sequence other than the fragment of the present invention. In this case, the antibodies or fragments thereof of the present invention can be removed using methods well known to those skilled in the art.

The culture may vary in the composition of the medium and the culture conditions depending on the type of the cells, and can be appropriately selected and controlled by those skilled in the art.

The antibody molecule may be accumulated in the cytoplasm of a cell, secreted from the cell, targeted by a suitable signal sequence to a periplasm or extracellular medium (supernatant), and labeled with a periplasmic or extracellular medium . It is also desirable to refold the engineered antibody molecules using methods well known to those of ordinary skill in the art and to have functional conformation.

The recovery of the polypeptide may vary depending on the characteristics of the produced polypeptide and the characteristics of the cells, and those skilled in the art can appropriately select and control the polypeptide.

The polypeptide may be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If a polypeptide is produced in a cell, it can be destroyed to release the protein as a first step. Particulate debris, host cells, or lysed fragments are removed, for example, by centrifugation or ultrafiltration. When the antibody is secreted into the medium, the supernatant from such an expression system is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. To inhibit proteolysis, a protease inhibitor, such as PMSF, may be included in any preceding step and antibiotics may be included to prevent the growth of contingent contaminants.

Antibodies prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, and the antibodies of the invention can be purified, preferably through affinity chromatography have.

The antibody or fragment thereof of the present invention specifically binds to EPRS, and thus is useful for diagnostic assays for detecting and quantifying EPRS proteins, for example, detecting EPRS expression in a specific cell, tissue, or serum.

Thus, the present invention provides an EPRS-specific detection method comprising contacting the antibody or fragment thereof with a sample and detecting the antibody or fragment thereof.

To " detect " the antibody or fragment thereof, the antibody or fragment thereof may generally be labeled with a detectable moiety.

See, for example, Current Protocols in Immunology, Volumes 1 and 2, 1991, Coligen et al., Ed. Wiley-Interscience, New York, N.Y., Pubs], using radioimmunoassay or fluorescent labeling. Radioactivity can be measured, for example, by scintillation counting, and fluorescence can be quantified using a fluorimeter.

Or various enzyme-substrate labels are available. Examples of such enzymatic labels are luciferase, luciferin, 2,3-dihydrophene, and the like, such as Fusarium luciferase and Bacillus luciferase (US Patent No. 4,737,456) Alkaline phosphatase,? -Galactosidase, glucoamylase, lysozyme, saccharide such as thalidomide, thalidomide, thalidomide, thalidomide, thalidomide, thalidomide, (Such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e. G., Free radicals and xanthine oxidases), lactoperoxidases, Oxydase, and the like. Techniques for conjugating an enzyme to an antibody are described, for example, in O'Sullivan et al., 1981, Methods for the Preparation of Enzyme-in-vivo Conjugates for Use in Enzyme Immunoassay, in Methods in Enzym. J. Langone & H. Van Vunakis, eds., Academic press, N. Y., 73: 147-166.

The label may be indirectly conjugated to the antibody using a variety of known techniques. For example, the antibody can be conjugated to biotin and any label belonging to the three broad categories mentioned above can be conjugated to avidin, or vice versa. Biotin selectively binds to avidin, and thus this label can be conjugated to the antibody in this indirect manner. Alternatively, to achieve an indirect conjugation of the label to the antibody, the antibody may be conjugated to a small hapten (e. G., Digoxin) and one of the different types of labels mentioned above may be conjugated to an anti- Hapten antibody (e. G., An anti-diphoshin antibody). Thus, indirect conjugation of the label to the antibody can be achieved.

The antibodies or fragments thereof of the invention can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays.

The antibody or fragment thereof of the present invention can be used in a diagnostic kit, that is, a packaged combination of reagents in predetermined amounts with a diagnostic kit, i.e., instructions for performing diagnostic analysis. In the case where the antibody is labeled with an enzyme, the kit may comprise a substrate and a cofactor required by the enzyme as a substrate precursor to provide chromophore or fluorophore. Other additives may also be included, such as stabilizers, buffers (e. G., Blocking buffer or lysis buffer), and the like. The relative amounts of the various reagents can be varied widely to provide a concentration in the solution of the reagent that sufficiently optimizes the sensitivity of the assay. The reagent may be provided as a generally lyophilized, dry powder comprising an excipient that will provide a reagent solution having an appropriate concentration upon dissolution.

The possibility of EPRS as a biomarker for diagnosis is confirmed by interferon gamma (IFN-γ) binding to the 3-UTR portion of mRNA of various regulatory proteins to form a complex, thereby regulating various inflammatory and immune responses (Sampath, P. et al., Noncanonical function of glutamylprolyltRNA synthetase: gene-specific silencing of translation, Cell 119, 195-208 (2004)). In addition, the protein synthesis process of VEGF (Vascular Endothelial Growth Factor A), an important factor in angiogenesis, has also been shown to be regulated by EPRS (Ray, PS et al. 457, 915-919 (2009)).

Thus, EPRS can be used as a diagnostic marker for the diagnosis of certain cancers, inflammatory diseases and immune diseases, progression of disease and evaluation of prognosis before and after treatment through detection. The diagnosis and prognostic evaluation of cancer, inflammatory diseases and immune diseases according to the present invention can be performed by detecting EPRS protein in a biological sample. Accordingly, the present invention provides a composition for diagnosing cancer, an inflammatory disease or an immunological disease comprising the antibody or fragment thereof of the present invention as an active ingredient.

Such biological samples include blood and other liquid samples of biological origin, biopsy specimens, solid tissue samples such as tissue culture, or cells derived therefrom. More specifically, it may be a tissue, an extract, a cell lysate, a whole blood, a plasma, a serum, a saliva, an ophthalmic solution, a cerebrospinal fluid, a sweat, a urine, a milk, a plural liquid, a synovial fluid, a 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 in detection. For example, it may include filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like. Further, nucleic acid and protein can be separated from the sample and used for detection.

The detection has been described above.

The type of the cancer is not particularly limited and examples thereof include breast cancer, colon cancer, lung cancer, small cell lung cancer, gastric cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, Ovarian cancer, ovarian cancer, rectal cancer, proximal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine cancer, thyroid cancer, Renal cell carcinoma, renal pelvic carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma , And preferably can be a carcinoma in which metastasis occurs.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising an antibody or fragment thereof as an effective ingredient.

The present invention also provides a pharmaceutical composition for preventing or treating immunological diseases comprising an antibody or a fragment thereof as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing or treating inflammation, comprising an antibody or fragment thereof as an active ingredient.

The composition of the present invention is characterized by containing the antibody or fragment thereof of the present invention as an active ingredient.

The pharmaceutical composition according to the present invention may comprise the antibody or fragment thereof of the present invention alone, or may further comprise at least one pharmaceutically acceptable carrier. The " pharmaceutically effective amount " as used herein refers to an amount exhibiting a further reaction than the negative control, preferably an amount sufficient to treat cancer.

The term " pharmaceutically acceptable " as used herein means a non-toxic composition which is physiologically acceptable and which, when administered to humans, does not inhibit the action of the active ingredient and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, .

In the pharmaceutical composition according to the present invention, the antibody or the fragment thereof may be administered in various formulations for oral administration and parenteral administration in clinical administration. In the case of formulation, usually used fillers, extenders, binders, wetting agents, , A surfactant, and the like.

Solid formulations for oral administration include tablets, patients, powders, granules, capsules, troches, and the like, which may contain one or more of the aryl derivatives of Formula 1, or a pharmaceutically acceptable salt thereof, At least one excipient, for example, starch, calcium carbonate, sucrose or lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions or syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, preservatives and the like are included .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.

The therapeutic compositions of the present invention may be formulated with any physiologically acceptable carrier, excipient or stabilizer (Remington: The Science and Practice of Pharmacy, l9th Edition, Alfonso, R., ed., Mack Publishing Co. )) And an antibody having a desired purity can be prepared in the form of a lyophilized cake or an aqueous solution for storage. Acceptable carriers, excipients or stabilizers are nontoxic to the recipient at the dosages and concentrations employed and include 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 dextrin; Chelating agents such as EDTA; Sugar alcohols such as mannitol or sorbitol; Salt-forming counterions such as sodium; And / or non-ionic surfactants such as tween, pluronics or polyethylene glycol (PEG).

The dose of the antibody or fragment of the present invention to the human body may vary depending on the patient's age, body weight, sex, dosage form, health condition, and disease severity, and is generally 0.01 to 100 mg / kg / Preferably from 0.1 to 20 mg / kg / week, more preferably from 5 to 10 mg / kg / week. It may also be administered at a fixed interval according to the judgment of a doctor or pharmacist.

The administration route of the composition of the present invention may be administered orally or parenterally by a known method such as injection or infusion by intravenous, intraperitoneal, intracerebral, subcutaneous, intramuscular, intravenous, intraarterial, intracerebral, or intralesional routes, Injection or injection by a sustained release system. Preferably, the antibody can be administered systemically.

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

In one embodiment of the present invention, a library is constructed in which a human VH3-23 / VL1g gene is skeletonized and a random sequence is inserted into a CDR, and a phage that selectively binds to EPRS is selected to isolate and purify the antibody. Respectively.

In another embodiment of the present invention, whether the purified antibody binds to EPRS was confirmed by the western blot method. As a result, it was confirmed that the antibody of the present invention binds to EPRS.

In another embodiment of the present invention, the affinity of the anti-EPRS scFv antibody of the present invention and EPRS was measured by surface resonance analysis. As a result, it was confirmed that the antibody of the present invention had an equilibrium dissociation constant of about 100 to 300 nM, indicating a relatively high affinity.

In another embodiment of the present invention, the cross reactivity of the anti-EPRS scFv antibody of the present invention was measured. As a result of measuring the reactivity of the antibody of the present invention with seven kinds of similar proteins including LRS, LUMINEX bead and EPRS, the antibodies of the present invention did not bind to WRS or HRS having other WHO domains except for EPRS It was confirmed to be very specific.

In another embodiment of the present invention, the use of the anti-EPRS scFv antibody of the present invention as a diagnostic antibody was tested. After coating the plate coated with the antibody of the present invention, the EPRS WHEP domains standard substance was diluted by concentration, reacted, and the binding was measured by ELISA. As a result, it was confirmed that antibody binding was measured in a concentration dependent manner on the EPRS WHEP domains standard substance, and it was confirmed that the antibody of the present invention can be used for diagnosis of cancer, inflammatory disease or immune disease.

Therefore, the antibody or fragment thereof of the present invention specifically binds to human EPRS and is not cross-reactive with other proteins including the same ARS family, so that detection and inhibition of EPRS can be performed. Therefore, EPRS detection and EPRS Or immunological disease diagnosis and treatment.

Figure 1 shows the results of a western blot experiment confirming whether the antibody of the present invention binds to the target protein EPRS.
FIG. 2 is a graph showing the binding affinity of anti-EPRS scFv Biocon-EP1 as an antibody of the present invention measured by surface plasmon resonance (SPR) (Kd: equilibrium dissociation constant, transverse axis: time (RU).
FIG. 3 is a graph showing the binding affinity of anti-EPRS scFv Biocon-EP2 of the present invention as a result of surface plasmon resonance (SPR) (Kd: equilibrium dissociation constant, transverse axis: time (RU).
FIG. 4 is a graph showing the binding affinity of the anti-EPRS scFv Biocon-EP3 antibody of the present invention measured by surface plasmon resonance (SPR) (Kd: equilibrium dissociation constant, transverse axis: time (sec) (RU).
5 is a graph showing the cross activity of the anti-EPRS scFv Biocon-EP1 antibody of the present invention measured by the Luminex Multiplex Assay method (longitudinal axis (FI): fluorescent intensity, CRS: tRNA synthetase, DRS: Aspartyl-tRNA synthetase, EPRS: Glutamyl-prolyl tRNA synthetase, HRS: Histidyl-tRNA synthetase, tRNA synthetase, WRS: tryptophanyl-tRNA synthetase, AIMP1: ARS-binding multifunctional protein 1, AIMP3: ARS (Aminoacyl-tRNA-synthetase-interacting multifunctional protein 3).
6 is a graph showing the cross activity of the anti-EPRS scFv Biocon-EP2 antibody of the present invention measured by the Luminex Multiplex Assay method (longitudinal axis (FI): fluorescent intensity, CRS: tRNA synthetase, DRS: Aspartyl-tRNA synthetase, EPRS: Glutamyl-prolyl tRNA synthetase, HRS: Histidyl-tRNA synthetase, tRNA synthetase, WRS: tryptophanyl-tRNA synthetase, AIMP1: ARS-binding multifunctional protein 1, AIMP3: ARS (Aminoacyl-tRNA-synthetase-interacting multifunctional protein 3).
FIG. 7 is a graph showing the cross activity of the anti-EPRS scFv Biocon-EP3 antibody of the present invention measured by the Luminex Multiplex Assay method (longitudinal axis (FI): fluorescent intensity, CRS: tRNA synthetase, DRS: Aspartyl-tRNA synthetase, EPRS: Glutamyl-prolyl tRNA synthetase, HRS: Histidyl-tRNA synthetase, tRNA synthetase, WRS: tryptophanyl-tRNA synthetase, AIMP1: ARS-binding multifunctional protein 1, AIMP3: ARS (Aminoacyl-tRNA-synthetase-interacting multifunctional protein 3).
FIG. 8 shows the results of an ELISA experiment (longitudinal axis: absorbance at 450 nm, abscissa: concentration of EPRS WHEP domains standard substance (ng / ml)) to confirm whether the antibody of the present invention can detect EPRS.

Hereinafter, the present invention will be described in detail with reference to examples.

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

≪ Example 1 >

scFv library construction and anti-EPRS scFv screening

<1-1> Construction of scFv library

The scFv library was constructed according to the method described in Korean Patent No. 10-0961392.

The library was designed by inserting a random sequence into the complementarity determining regions (CDRs) of human VH3-23 / VL1g skeleton and using a plasmid vector having the beta lactamase gene as a selection marker The pFDV plasmid vector was prepared by introducing restriction enzyme cleavage sequences immediately after the leader sequence of the beta lactamase gene, inserted into the scFv gene library, transfected into Escherichia coli, and cultured to construct a library. Through this process it is possible to improve the quality of the library by removing most of the scFv gene sequences with abnormal stop codons or frame shifts in the library. After removing the error-eliminated sequences from the cultured library, the scFv gene library was amplified by polymerase chain reaction and inserted into the pComb3X phagemid vector, and Escherichia coli of E. coli ER2537 strain was transfected to obtain a final library . The library was incubated in 400 mL of SB (super broth) medium containing carbenicillin, and when the absorbance at 600 nm was 0.5, 1013 CFU of VCSM13 supplemental phage was added and stirred at 80 rpm for 1 hour at 37 DEG C Lt; / RTI &gt; The final 70 μg / mL of kanamycin antibiotics were added and incubated at 30 ° C and 200 rpm overnight to produce scaffolded phage. On the next morning, the culture was centrifuged and the phage in the culture was precipitated by addition of 4% polyethylene glycol-8000 and 3% sodium chloride. The precipitated phage was dissolved in 50 mL of PBS buffer, precipitated again in the same manner as described above, and finally dissolved in 2 mL of PBS buffer solution. This was centrifuged to remove impurities and a scaffold phage library was obtained. In general, the final phage library contains phage particles of 1013 CFU / mL or more.

<1-2> Selection of EPRS scFv

EPRS WHEP domain antigen was added to the immunotube at a concentration of 10 μg / ml. After the protein was adsorbed on the surface of the test tube for 1 hour, a 3% solution of powdered milk was added to the test tube to protect the surface that did not absorb EPRS. After the test tube was emptied, 1012 CFU antibody phage library dispersed in a 3% powdered milk solution was added to bind the antigen. The non - specifically bound phage was rinsed 3 times with TBST (tris buffered saline - tween 20) solution, and the remaining antigen - specific retention antibody was eluted with 100 mM triethylamine solution.

The eluted phages were neutralized with a 1.0 M Tris-HCl buffer (pH 7.8) and then infected with ER2537 E. coli at 37 DEG C for 1 hour. The infected E. coli was applied to LB (Luria-Bertani) agar medium containing carbenicillin And cultured at 37 ° C. The next day, the cultured Escherichia coli was suspended in 3 mL of SB (super broth) -carbenicillin culture solution, 15% glycerol was added, a portion was stored at -80 DEG C, and 50 microliters of the remaining was added to 20 mL of SB- -2% glucose solution and incubated at 37 ° C. When the absorbance of the 600 nm light of the culture solution becomes 0.5, the bacteria are separated by centrifugation, suspended in 20 mL of SB-carbenicillin culture solution, and then 1012 PFU (plaque forming unit) VCSM13 auxiliary phage is added and stirred slowly And cultured at 37 ° C.

One hour later, 70 ug / ml of kanamycin was added and incubated overnight (250 rpm) with rapid stirring at 30 캜. The next day, the culture was centrifuged and antigen-specific clones were concentrated by repeating the above panning procedure using 1 mL of supernatant containing phage particles as a library.

&Lt; Example 2 >

Anti-EPRS scFv antibody expression and purification

After repeated panning about 3-4 times, E. coli containing the antibody gene was coated on an LB agar medium containing carbenicillin and cultured to obtain single colonies, which were inoculated and cultured in 200 μL SB-carbenicillin solution And then expressed in IPTG to express the scFv protein in the periplasm of E. coli. After suspending E. coli in 40 μL of 1 × TES (50 mM Tris, 1 mM EDTA, 20% Sucrose, pH 8.0) solution, add 60 μL of 0.2X TES solution to the mixture and incubate at 4 ° C for 30 min. And the periplasm was extracted with the supernatant.

&Lt; 2-1 > Expression of scFv antibody selected for EPRS

The scFv-positive single colony clones for the selected EPRS were cultured in 5 ml of SB medium containing carbenicillin (Bactotrytone 30 g, yeast extract 20 g, MOPS buffer 10 g / L), seed culture was started, The scFv protein was expressed in periplasm of Escherichia coli by incubating overnight at 30 ° C. with 1 mM IPTG. The next day, E. coli obtained by centrifugation was suspended in 1 × TES buffer (50 mM Tris, 1 mM EDTA, 20% sucrose, pH 8.0) and then 0.2 × TES was added 1.5 times and the mixture was centrifuged to extract periplasm .

&Lt; 2-2 > Purified scFv antibody purified

To the scFv antibody extracted from the periplasm, 5 mM MgSO 4 was added and the mixture was mixed with Ni-NTA beads previously equilibrated with PBS. After stirring for 1 hour in a refrigerator, the resultant was bound to Ni-bead and affinity chromatography was performed Proteins were washed away with PBS. After washing with a buffer containing 5 mM Imidazole, the bound scFv antibody was eluted with 200 mM Imidazole buffer. The eluted antibody was dialyzed and its purity was confirmed by electrophoresis. The protein amount was quantified by BCA method, and the amount of the purified antibody was recorded.

<2-3> Immunoblotting and sequencing

The scFv antibody extracted from the periplasm was used to confirm whether the scFv antibody binds to the originally expressed EPRS in human cells using an immunoblotting (western blot) technique. 50 ug of Hela cell lysate was electrophoresed through SDS PAGE, transferred to Nitrocellulos membrane by wet transfer method, blocked with 3% skim milk, and combined with scFv antibody. For detection, anti-HA secondary antibody conjugated with horseradish peroxidase (HRP) was reacted with bound scFv, and then film-sensitized with ECL reagent in the dark. A band corresponding to the size of the EPRS was identified as compared to the exposed standard molecular marker.

The antigen-specific antibody clones identified from the above were incubated overnight in 10 ml of SB medium containing carbenicillin, plasmid DNA was extracted using a plasmid miniprep kit, the sequence was analyzed by Capillary sequencing service (Macrogen Co), and Kabat protein The CDR sequence was analyzed based on the seqeunce database and IMGT (R) (the international ImMunoGeneTics information system®) analysis method.

As a result, the number of scFvs bound to the EPRS antigen was three in total, and the nucleotide sequences thereof were found to be SEQ ID NOS: 14, 28 and 42.

&Lt; Example 3 >

Determination of affinity of anti-EPRS scFv antibody

The binding affinity of the antibody of the present invention to the EPRS WEHP domain antigen was measured using ProteOnTMXPR36 surface plasmon resonance (SPR) biosensor (Bio-Rad).

Specifically, about 10 ug / ml of EPRS antigen was reacted with about 2,000 to 4,000 response units on a GLC chip (Bio-Rad 6 X 6 sensor chip, Compact capacity amine coupling for protein-protein interactions) according to the manufacturer's instructions. , 30 ul of the purified scFv antibody (250-30 nM) of the present invention diluted to various concentrations by using PBS was injected into the chip at 25 ° C at a rate of 50 μl / min to quantify the interaction with the antigen . The surface of the chip was regenerated with 0.85% phosphoric acid and the combined speed and dissociation rate were calculated using ProteOn Manager software and the equilibrium dissociation constant (KD) was calculated as dissociation rate /

As a result, as shown in [Figure 2-4], the antibody of the present invention had a maximum value of KD 10 nM, confirming that it had a very high EPRS affinity.

<Example 4>

Measurement of anti-EPRS scFv antibody cross activity

The cross reactivity of the antibody of the present invention with EPRS and other ARS family proteins was measured using Luminex bead to determine whether the scFv antibody was reactive with other antigens.

&Lt; 4-1 > Production of Luminex beads combined with each protein

Coupling was performed according to the experimental procedure of Bio-rad's Amine coupling kit to bind the amine residue of the protein to each bead of different Code No. Each bead of 1 x 106 was transferred to a 96-well filter plate, washed with a vacuum manifold in a buffer of activation, and resuspended in 50 mg / ml S-NHS (N-hydroxysulfosuccinimide) and 50 mg / ml EDAC [3-dimethylaminopropyl] carbodiimide) was added and the mixture was allowed to react at room temperature for 20 minutes. Each bead was washed with PBS and then added with 10 ug of purified recombinant ARS antigen (CRS, DRS, EPRS WHEP domain, HRS, WRS, AIMP1 (p43) and AIMP3 And reacted for 2 hours. The beads connected to each ARS were washed twice with PBS, blocked with buffer, and reacted at room temperature for 30 minutes to block unbound residues. After washing twice with PBS, the beads were suspended again in 100 μl PBS, Respectively. The number of combined beads was measured with a hemocytometer.

<4-2> Cross activity measurement using Multiplex Assay

The antibody of the present invention extracted from the periplasm was first diluted in a 1: 400 concentration of a sample dilution (PBST + 2% BSA), diluted twice with a sample dilution buffer in a 96 well filter plate, Only sample dilution was added to blank wells. Each bead combined with ARS was placed into a bead mix tube to be 2,000 beads per well, and a volume of sample dilution was added to each well in an amount of 50 ul per well. bead mix solution was mixed well and then divided into each well and reacted in a dark room for 1 hour. After completion of the reaction, the cells were washed three times with PBS (200 μl / well) using a vacuum manifold, and then 50 μl of anti- (HA) -biotin secondary antibody was further added thereto for 1 hr in the dark. The beads bound to the secondary antibody were washed three times with PBS and stained with 2 μg / ml of SA-PE (Streptavidin-Phycoerythrin) for fluorescent labeling. The beads were reacted in a dark room for 30 minutes, washed with PBS 3 times, Lt; / RTI &gt; Fluorescence-labeled beads were analyzed for fluorescence intensities using a Bio-Plex (Luminex) 200 instrument and a Bio-Plex manager program to confirm the binding of antibodies to each ARS.

As a result, as shown in FIG. 5 to FIG. 7, it was confirmed that the antibody of the present invention did not react with other ARS family proteins except EPRS at all concentrations.

Therefore, it was confirmed that the antibody of the present invention was not cross-reactive with other proteins.

&Lt; Example 5 >

Sandwich ELISA pairing test using purified anti-EPRS scFv antibody

To investigate the usefulness of the antibody of the present invention as a diagnostic antibody, an EPRS sandwich ELISA pairing test was performed. In the ELISA construct, a capture antibody was used as an antibody of the present invention and a detection antibody was pairing with a rabbit polyclonal antibody To confirm that a quantitative curve for the EPRS standard material is produced.

First, the purified antibody of the present invention was diluted in a coating buffer (0.1 M sodium carbonate pH 9.0) and dispensed in an amount of 100 to 400 ng per well in an ELISA plate, followed by standing at room temperature for 3 hours. After washing 3 times with PBST, 350 μl of PBST containing 3% skim milk was added, followed by blocking at room temperature for 1 hour and then washing with PBST three times. The purified recombinant EPRS standard substance was diluted twice in duplicate in the sample dilution solution in a plate well coated with antibody, and 100 μl of each dilution was added to each well and reacted at room temperature for 1 hour. After the reaction, the plate was washed three times with PBST, and then 100 μl of the diluted detection antibody (4 μg / ml) was added, followed by further reaction at room temperature for 1 hour. For detection, the plate was washed three times with PBST, and HRP-conjugated anti-rabbit IgG was added thereto and allowed to react at room temperature for 1 hour. To evaluate the color reaction by HRP, the plate was washed three times with PBST, and then 50 μl of TMB solution, which is a HRP substrate, was added thereto. The color reaction was observed for 10 minutes and 50 μl of 2N sulfuric acid was added to stop the color reaction. The absorbance was measured.

As a result, as shown in FIG. 8, it was confirmed that a corresponding standard line was drawn from a concentration of less than 50 ng / ml for the recombinant EPRS WHEP domain reference material. Thus, it was confirmed that the antibody of the present invention can be used for diagnosis of cancer and immune diseases.

As described above, the antibody or fragment thereof of the present invention specifically binds to human EPRS and can not detect cross-reactivity with other proteins including the same ARS family, so that EPRS detection and inhibition are possible. Therefore, EPRS detection and EPRS , An inflammatory disease or an immune disease diagnosis and treatment, which is highly likely to be used industrially.

<110> Ewha University-Industry Collaboration Foundation          Medicinal Bioconvergence Research Center <120> anti-EPRS monoclonal antibodies and uses thereof <130> NP14-0098 <160> 42 <170> Kopatentin 2.0 <210> 1 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> VL-CDR1 of Biocon EP1 <400> 1 Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Ala Val Asn   1 5 10 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VL-CDR2 of Biocon EP1 <400> 2 Ala Asn Ser His Arg Pro Ser   1 5 <210> 3 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VL-CDR3 of Biocon EP1 <400> 3 Gly Thr Trp Asp Tyr Ser Leu Asn Gly Tyr Val   1 5 10 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> VH-CDR1 of Biocon EP1 <400> 4 Asp Tyr Asp Met Ser   1 5 <210> 5 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH-CDR2 of Biocon EP1 <400> 5 Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Mar   1 5 10 15 Gly     <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VH-CDR3 of Biocon EP1 <400> 6 Asn Arg Phe Val Phe Asp Tyr   1 5 <210> 7 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding VL-CDR1 of Biocon EP1 <400> 7 agtggctctt catctaatat tggcaataat gctgtcaac 39 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding VL-CDR2 of Biocon EP1 <400> 8 gctaatagtc atcggccaag c 21 <210> 9 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding VL-CDR3 of Biocon EP1 <400> 9 ggtacttggg attatagcct gaatggttat gtc 33 <210> 10 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding VH-CDR1 of Biocon EP1 <400> 10 gattatgata tgagc 15 <210> 11 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding VH-CDR2 of Biocon EP1 <400> 11 gcgatctctt ctggtggtgg taatacatat tacgctgatt ctgtaaaagg t 51 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding VH-CDR3 of Biocon EP1 <400> 12 aatcgttttg tgttcgacta c 21 <210> 13 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> scFv of Biocon EP1 <400> 13 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 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Gly Ile Tyr His Gly Gly 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 Thr Arg Thr Leu 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 Ser Gly Ser Ser Ser Asn Ile 145 150 155 160 Gly Asn Asn Ala Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro                 165 170 175 Lys Leu Leu Ile Tyr Ala Asn Ser His 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 Tyr Ser Leu Asn Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val 225 230 235 240 Leu     <210> 14 <211> 723 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding scFv of Biocon EP1 <400> 14 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgggactc 60 tcctgtgcag cctctggatt cacctttagc gattatgata tgagctgggt ccgccgggct 120 ccagggaagg ggctggagtg ggtctcagcg atctcttctg gtggtggtaa tacatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagaaatcgt 300 tttgtgttcg actactgggg ccagggtaca ctggtcaccg tgagctcagg tggaggcggt 360 tcaggcggag gtggatccgg cggtggcgga tcgcagtctg tgctgactca gccaccctca 420 gcgtctggga cccccgggca gagggtcacc atctcttgta gtggctcttc atctaatatt 480 ggcaataatg ctgtcaactg gtaccagcag ctcccaggaa cggcccccaa actcctcatc 540 tatgctaata gtcatcggcc aagcggggtc cctgaccgat tctctggctc caagtctggc 600 acctcagcct ccctggccat cagtgggctc cggtccgagg atgaggctga ttattactgt 660 ggtacttggg attatagcct gaatggttat gtcttcggcg gaggcaccaa gctgacggtc 720 cta 723 <210> 15 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> VL-CDR1 of Biocon EP2 <400> 15 Thr Gly Ser Ser Ser Asn Ile Gly Asn Asn Ala Val Ser   1 5 10 <210> 16 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VL-CDR2 of Biocon EP2 <400> 16 Ala Asn Ser His Arg Pro Ser   1 5 <210> 17 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VL-CDR3 of Biocon EP2 <400> 17 Gly Thr Trp Asp Ala Ser Leu Ser Gly Tyr Val   1 5 10 <210> 18 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> VH-CDR1 of Biocon EP2 <400> 18 Asn Tyr Ala Met Ser   1 5 <210> 19 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH-CDR2 of Biocon EP2 <400> 19 Gly Ile Tyr His Gly Gly Gly Asn Lys Tyr Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly     <210> 20 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VH-CDR3 of Biocon EP2 <400> 20 Thr Arg Thr Leu Phe Asp Tyr   1 5 <210> 21 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding VL-CDR1 of Biocon EP2 <400> 21 actggctctt catctaatat tggcaataat gctgtctcc 39 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding VL-CDR2 of Biocon EP2 <400> 22 gctaatagtc atcggccaag c 21 <210> 23 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding VL-CDR3 of Biocon EP2 <400> 23 ggtacttggg atgctagcct gagtggttat gtc 33 <210> 24 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding VH-CDR1 of Biocon EP2 <400> 24 aactatgcta tgagc 15 <210> 25 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding VH-CDR2 of Biocon EP2 <400> 25 gggatctatc atggtggtgg taataaatat tacgctgatt ctgtaaaagg t 51 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding VH-CDR3 of Biocon EP2 <400> 26 acgaggacgc ttttcgacta c 21 <210> 27 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> scFv of Biocon EP2 <400> 27 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 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Gly Ile Tyr His Gly Gly 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 Thr Arg Thr Leu 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 Ala Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro                 165 170 175 Lys Leu Leu Ile Tyr Ala Asn Ser His 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 Ala Ser Leu Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val 225 230 235 240 Leu     <210> 28 <211> 723 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding scFv of Biocon EP2 <400> 28 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc aactatgcta tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcaggg atctatcatg gtggtggtaa taaatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagaacgagg 300 acgcttttcg actactgggg ccagggtaca ctggtcaccg tgagctcagg tggaggcggt 360 tcaggcggag gtggatccgg cggtggcgga tcgcagtctg tgctgactca gccaccctca 420 gcgtctggga cccccgggca gagggtcacc atctcttgta ctggctcttc atctaatatt 480 ggcaataatg ctgtctcctg gtaccagcag ctcccaggaa cggcccccaa actcctcatc 540 tatgctaata gtcatcggcc aagcggggtc cctgaccgat tctctggctc caagtctggc 600 acctcagcct ccctggccat cagtgggctc cggtccgagg atgaggctga ttattactgt 660 ggtacttggg atgctagcct gagtggttat gtcttcggcg gaggcaccaa gctgacggtc 720 cta 723 <210> 29 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> VL-CDR1 of Biocon EP3 <400> 29 Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Ala Val Asn   1 5 10 <210> 30 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VL-CDR2 of Biocon EP3 <400> 30 Ser Asn Ser His Arg Pro Ser   1 5 <210> 31 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VL-CDR3 of Biocon EP3 <400> 31 Gly Ser Trp Asp Ala Ser Leu Asn Gly Tyr Val   1 5 10 <210> 32 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> VH-CDR1 of Biocon EP3 <400> 32 Ser Tyr Tyr Met Ser   1 5 <210> 33 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH-CDR2 of Biocon EP3 <400> 33 Leu Ile Ser Pro Gly Ser Gly Ser Ile Tyr Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly     <210> 34 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VH-CDR3 of Biocon EP3 <400> 34 Thr Gly Trp Leu Phe Asp Tyr   1 5 <210> 35 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding VL-CDR1 of Biocon EP3 <400> 35 agtggctctt catctaatat tggcaataat gctgtcaac 39 <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding VL-CDR2 of Biocon EP3 <400> 36 tctaatagtc atcggccaag c 21 <210> 37 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding VL-CDR3 of Biocon EP3 <400> 37 ggttcttggg atgctagcct gaatggttat gtc 33 <210> 38 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding VH-CDR1 of Biocon EP3 <400> 38 agttattata tgagc 15 <210> 39 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding VH-CDR2 of Biocon EP3 <400> 39 ttgatctctc ctggtagtgg tagtatatat tacgctgatt ctgtaaaagg t 51 <210> 40 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding VH-CDR3 of Biocon EP3 <400> 40 actggttggc ttttcgacta c 21 <210> 41 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> scFv of Biocon EP3 <400> 41 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 Val Ser Gly Phe Thr Phe Ser Ser Tyr              20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Leu Ile Ser Pro Gly Ser Gly Ser Ile 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 Thr Gly Trp Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Asn Gly 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 Ser Gly Ser Ser Ser Asn Ile 145 150 155 160 Gly Asn Asn Ala Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro                 165 170 175 Lys Leu Leu Ile Tyr Ser Asn Ser His 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 Asn Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val 225 230 235 240 Leu     <210> 42 <211> 723 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding scFv of Biocon EP3 <400> 42 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgaggctc 60 tcctgtgcag tctctgggtt cacctttagc agttattata tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcattg atctctcctg gtagtggtag tatatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagaactggt 300 tggcttttcg actactgggg ccagggtaca ctggtcaccg tgaactcagg tggaggcggt 360 tcaggcggag gtggatccgg cggtggcgga tcgcagtctg tgctgactca gccaccctca 420 gcgtctggga cccccgggca gagggtcacc atctcttgta gtggctcttc atctaatatt 480 ggcaataatg ctgtcaactg gtaccagcag ctcccaggaa cggcccccaa actcctcatc 540 tattctaata gtcatcggcc aagcggggtc cctgaccgat tctctggctc caagtctggc 600 acctcagcct ccctggccat cagtgggctc cggtccgagg atgaggctga ttattactgt 660 ggttcttggg atgctagcct gaatggttat gtcttcggcg gaggcaccaa gctgacggtc 720 cta 723

Claims (16)

i) a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 1, a complementarity determining region (CDR) L2 including the amino acid sequence represented by SEQ ID NO: 2, and an amino acid sequence represented by SEQ ID NO: 3 An antibody light chain variable region (VL) comprising the complementarity determining region (CDR) L3 and a complementary crystal region (CDR) H1 comprising the amino acid sequence represented by SEQ ID NO: 4, a complementary region comprising the amino acid sequence represented by SEQ ID NO: 5 An antibody heavy chain variable region (VH) comprising a crystal region (CDR) H2, a complementary crystal region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 6;
ii) a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 15, a complementarity determining region (CDR) L2 including the amino acid sequence represented by SEQ ID NO: 16, and an amino acid sequence represented by SEQ ID NO: An antibody light chain variable region (VL) comprising the complementary crystal region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 18, a complementary crystal region (CDR) H1 comprising the amino acid sequence represented by SEQ ID NO: 18, An antibody heavy chain variable region (VH) comprising a crystal region (CDR) H2, a complementary crystal region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 20; And
iii) a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 29, a complementarity determining region (CDR) L2 including the amino acid sequence represented by SEQ ID NO: 30, and an amino acid sequence represented by SEQ ID NO: 31 An antibody light chain variable region (VL) comprising the complementary crystal region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 32 and a complementary crystal region (CDR) H1 comprising the amino acid sequence represented by SEQ ID NO: An antibody heavy chain variable region (VH) comprising a crystal region (CDR) H2, a complementary crystal region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 34;
Lt; RTI ID = 0.0 &gt; EPRS. &Lt; / RTI &gt;
2. The method of claim 1,
i) an antibody comprising a 132 to 241 amino acid sequence of SEQ ID NO: 13 as a light chain variable region and a 1 to 116 amino acid sequence of SEQ ID NO: 13 as a heavy chain variable region;
ii) an antibody comprising a 132 to 241 amino acid sequence of SEQ ID NO: 27 as a light chain variable region and a 1 to 116 amino acid sequence of SEQ ID NO: 27 as a heavy chain variable region; And
iii) an antibody comprising the amino acid sequence from position 132 to position 241 of SEQ ID NO: 41 as the light chain variable region and the amino acid sequence from position 1 to 116 of SEQ ID NO: 41 as the heavy chain variable region;
&Lt; / RTI &gt; wherein the antibody is an antibody selected from the group consisting of: &lt; RTI ID = 0.0 &gt;
The antibody or fragment thereof according to claim 1, wherein the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 27 and SEQ ID NO: 41. The antibody or fragment thereof according to claim 1, wherein the fragment is a fragment selected from the group consisting of diabodies, Fab, Fab ', F (ab) 2, F (ab') 2, Fv and scFv.
A polynucleotide encoding the antibody of claim 1 or a fragment thereof.
6. The polynucleotide according to claim 5, wherein the polynucleotide is
i) a polynucleotide represented by SEQ ID NO: 7 to 12;
ii) a polynucleotide represented by SEQ ID NOs: 21 to 26; And
iii) a polynucleotide represented by SEQ ID NO: 35 to 40;
&Lt; RTI ID = 0.0 &gt; polynucleotides &lt; / RTI &gt;
A vector comprising the polynucleotide of claim 5.
7. A cell comprising the vector of claim 7.
Culturing the cells of claim 8 under a condition that the polynucleotide is expressed to produce a polypeptide comprising a light chain and a heavy chain variable region and recovering the polypeptide from the cell or a culture medium in which the polypeptide is cultured A method of producing an antibody or fragment thereof that binds to EPRS. A method for detecting an EPRS, comprising contacting the antibody of claim 1 or a fragment thereof with a sample, and detecting the antibody or fragment thereof.
A pharmaceutical composition for preventing or treating cancer comprising the antibody of claim 1 or an antibody fragment thereof as an active ingredient.
A cancer diagnostic composition comprising the antibody of claim 1 or a fragment thereof as an active ingredient.
A pharmaceutical composition for preventing or treating an immunological disease comprising the antibody of claim 1 or an antibody fragment thereof as an active ingredient.
A composition for diagnosing an immune disease comprising the antibody of claim 1 or an antibody fragment thereof as an active ingredient.
A pharmaceutical composition for preventing or treating an inflammatory disease comprising the antibody of claim 1 or an antibody fragment thereof as an active ingredient.
A composition for diagnosing an inflammatory disease comprising the antibody of claim 1 or an antibody fragment thereof as an active ingredient.

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