KR20230015245A - Anti-HLA-DP monoclonal antibody and use thereof - Google Patents

Abstract

The present invention relates to an anti-HLA-DP monoclonal antibody which binds to HLA-DP with high affinity and specificity, and a use thereof. The antibody binds to HLA-DP with high affinity without cross-reaction with HLA-DQ or HLA-DR, and thus can be used for various purposes in in vitro cell-based experiments and in vivo efficacy experiments including immunoblotting and flow cytometry.

Description

Anti-HLA-DP monoclonal antibody and use thereof {Anti-HLA-DP monoclonal antibody and use thereof}

The present invention relates to anti-HLA-DP monoclonal antibodies and uses thereof, and more particularly, to anti-HLA-DP monoclonal antibodies binding to HLA-DP with high affinity and specificity and uses thereof.

The major histocompatibility complex (MHC) is a surface protein expressed on the surface of nucleated cells, and MHC is characterized by polygenicity and polymorphism. The human leukocyte antigen (HLA) gene is a gene encoding human MHC, located on chromosome 6, and the HLA complex is inherited from each parent according to Mendelian inheritance and co-dominant expression on the cell surface. HLA complexes are classified into Class I and Class II, and Class I and Class II molecules are structurally different. HLA Class I molecules exist in the form bound to β-microglobulin chains, and include HLA-A, -B, and -C. HLA class II is composed of α and β chains combined, and includes HLA-DR, -DP, and -DQ. HLA genes composed in this way have the most diversity among human genes, so the HLA haplotype composed of a combination of HLA genes plays a role in distinguishing self from non-self in the immune response. In the immune response, the HLA complex plays a role in inducing an immune response by presenting antigens to T lymphocytes, which are key cells of the immune response.Therefore, in the study of the immune response, research on the HLA complex is a key research field, and various studies are being conducted. However, research on HLA Class II has been focused on HLA-DR, and research on HLA-DP has been rapidly increasing recently.

On the other hand, HLA-DP genes include HLA-DPA1, -DPB1, -DPA2, -DPA3 and DPB2. The HLA-DP gene is expressed by HLA-DPA1 and HLA-DPB1, and HLA-DPA2, -DPA3, and -DPB2 are pseudogenes that are not actually expressed (HLA-DP related pseudogenes). HLA-DP is expressed on the cell surface in the form of a combination of DPA1 (alpha chain) and DPB1 (beta chain). A peptide of about 9 amino acids is loaded onto the HLA-DP binding site and presented to antigen presenting cells (APCs). In general, peptides presented by HLA-DP in HLA class II are exogenous antigens and CD4+ T cells recognize and stimulate peptide-HLA complexes. According to the IMGT/HLA database, as of June 2021, 2, 2015, 107, 1106, 143 and 1303 proteins are known for DRA, DRB1, DPA1, DPB1, DQA1 and DQB1, respectively. Thus, HLA-DP has the least polymorphism among the three HLA class II molecules. HLA-DP is genetically associated with various diseases, including autoimmune diseases, hepatitis, myeloid diseases, and organ transplantation. Autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, diabetes, Graves' disease, chronic beryllium disease, juvenile chronic arthritis, sarcoidosis, and atopic myelitis. ), polyangiitis, and granulomatosis have been reported (Diabetes　2010 Aug;　59(8):　2055-2062, Clin Rheumatol 2018 Jul;37(7):1799-1805. Scientific Reports 2017 ;7: 39757).

The crystal structure of the HLA-DP protein was confirmed in 2010, and recently, studies on the association between peptides and disease loading for HLA-DP have been conducted, but HLA-DP is as large as other HLA class II molecules. It has not been extensively studied. For the study of HLA and disease mechanisms, specific isolation, detection, and purification of HLA-DP molecules are required. In particular, a large amount of antibody is required to purify a large amount of HLA-DP molecules. Therefore, the present inventors have made diligent efforts to develop an effective antibody capable of specifically detecting HLA-DP, and as a result, a monoclonal hybridoma cell line producing an antibody specific to HLA-DPA1 according to the present invention was constructed, The present invention was completed by confirming that the cell line could produce an antibody with improved specificity compared to the conventional HLA-DPA1.

Diabetes　2010 Aug; 59(8):　2055-2062, Clin Rheumatol 2018 Jul;37(7):1799-1805 Scientific Reports 2017; 7: 39757

The present invention is to provide an antibody that can specifically bind to HLA-DP and uses thereof.

In order to achieve the above object, the present invention provides a heavy chain variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3; And / or a light chain variable region comprising CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 6; .

In the present invention, the antibody or antigen-binding fragment thereof is characterized in that it comprises a heavy chain variable region of SEQ ID NO: 7.

In the present invention, the antibody or antigen-binding fragment thereof is characterized in that it comprises a light chain variable region of SEQ ID NO: 8.

The present invention also provides nucleic acids encoding the antibodies or antigen-binding fragments thereof.

The present invention also provides a cell transformed with the nucleic acid or a vector containing the nucleic acid.

The present invention also provides a method for preparing an antibody or antigen-binding fragment thereof that specifically binds to HLA-DP, comprising the following steps:

(a) culturing the cells; and

(b) recovering the antibody or antigen-binding fragment thereof from the cultured cells.

Since the antibody binding to HLA-DP according to the present invention binds to HLA-DP with high affinity without cross-reaction with HLA-DQ or HLA-DR, in vitro cell-based experiments including immunoblotting and flow cytometry, in Not only can it be used for various purposes in in vivo efficacy experiments, but it will also be able to be used in various ways to study the pathogenesis of various immune diseases mediated by HLA-DP.

1 is a schematic diagram showing a limiting dilution method.
Figure 2 is a schematic diagram of hybridoma cell production for monoclonal antibody production.
Figure 3 shows the nucleotide sequence of HLA-DPA1, the codon-optimized sequence for cloning, and the amino acid sequence accordingly.
Figure 4 shows the ELISA results of 24 hybridoma clones showing the highest titer among 480 hybridoma clones. Except for #7, it was positive for the HLA-DPA1 antigen. P indicates commercial HLA-DPA1 antibody (NB-A3) as a positive control and N indicates PBS as a negative control.
5 is an ELISA result for HLAα chain antigen. P indicates commercial HLA-DPA1 antibody (NB-A3) as a positive control and N indicates PBS as a negative control. (A) For the DRA1 antigen, all clones showed a similar degree of binding to the blank. (B) All clones were confirmed to be positive for the DPA1 antigen, and the 5 most effective clones among them were marked in yellow. (C) All clones were confirmed negative for the DQA1 antigen.
6 is a flow cytometry analysis result of 23 hybridoma clones confirmed positive by ELISA. The unstained control without the addition of antibody is shown in gray, the negative control with the addition of only the secondary antibody is shown in black, and the positive control with the addition of a commercial HLA-DPA1 antibody (NB-A3) is shown in light blue. 17 clones were identified as negative and marked in blue, and 6 clones were identified as positive and marked in red.
7 is a schematic diagram of screening monoclonal antibodies.
Figure 8 shows the hybridoma limiting dilution method for HLA-DP, and the limiting dilution process was performed by flow cytometry. (a) is the limiting dilution result of clone #13, and 13-4-1-26-4 and 13-4-1-26-14 were selected as final clones. (b) is the limiting dilution result of clone #23, and 23-6-46-26 was selected as the final clone.
9 is a result of flow cytometry showing the binding affinity of the final clone. The unstained control without the addition of antibody is shown in gray, the negative control with the addition of only the secondary antibody is shown in black, and the positive control with the addition of a commercial HLA-DPA1 antibody (NB-A3) is shown in light blue. The top three clones selected as final clones are marked in red, and the unselected clones are marked in blue.
10 is a result of confirming the binding affinity of the purified antibody to human PBMC in order to confirm clinical applicability. The unstained control with no antibody added is shown in gray, the negative control with only secondary antibody added is shown in black, and the positive control with the addition of a commercial HLA-DPA1 antibody (NB-A3) is shown in orange. 13-4-1-26-4 is indicated by a red line, 13-4-1-26-14 by a green line, and 23-6-46-26 by a blue line.
Figure 11 shows the results of the binding affinity test of the 13-4-1-26-14 clone, and quantitatively compares the binding affinity of the anti-HLA-DPA1 antibody with that of commercially available antibodies using prepared HLA-transfected cells. did Controls without antibody are shown in gray, controls transfected with empty vector are shown in black, and HLA-transfected cells are shown in red, orange and green for HLA DR, HLA-DP and HLA-DQ, respectively.
12 shows the results of the isotyping test of the heavy and light chains of the antibody produced from the final hybridoma clone. As a result of the isotyping ELISA test of the monoclonal antibody, all three monoclonal antibodies were positive for the IgG2a heavy chain (upper gray). , all light chains were identified as kappa chains (bottom grey).
Figure 13 shows the results of homology sequence analysis of the heavy chain (top) and light chain (bottom) variable regions of the anti-HLA-DP monoclonal antibody.

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

In the present invention, a hybridoma cell capable of producing an antibody against HLA-DP is prepared by synthesizing the gene of HLA-DPA1 and using it as an immunogen, and a hybridoma capable of producing an effective monoclonal antibody from the hybridoma cell. Cells were selected. Finally, the monoclonal antibody produced from the selected hybridoma cells bound to HLA-DP with higher binding affinity than commercially available antibodies, and as a result of confirmation using human cell lines expressing HLA-DR, DP, and DQ, HLA It did not bind to -DR or HLA-DQ, but it was confirmed to bind to HLA-DP with very high specificity. Accordingly, as a result of analyzing the variable domain sequence of the monoclonal antibody, the sequence was found to be a novel sequence that has not been previously reported.

Accordingly, in one aspect, the present invention provides a heavy chain variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3; And / or a light chain variable region comprising CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 6; .

SEQ ID NO: 1: heavy chain CDR1

GFTFSSYG

SEQ ID NO: 2: heavy chain CDR2

ISRGDSYT

SEQ ID NO: 3: heavy chain CDR3

ARTFAY

SEQ ID NO: 4: light chain CDR1

KSVSTSGYSY

SEQ ID NO: 5: light chain CDR2

LVS

SEQ ID NO: 6: light chain CDR3

QHIRELTR

In the present invention, the antibody or antigen-binding fragment thereof may be an antibody or antigen-binding fragment thereof comprising the heavy chain variable region of SEQ ID NO: 7, but is not limited thereto.

SEQ ID NO: 7: heavy chain variable region

VQLQESGGDLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLEWVATISRGDSYTYYPDSVKGRFAISRDNAKNTLYLQMSSLKSEDTATYYCARTFAYWGQGTLVTVSA

In the present invention, the antibody or antigen-binding fragment thereof may be an antibody or antigen-binding fragment thereof, including the light chain variable region of SEQ ID NO: 8, but is not limited thereto.

SEQ ID NO: 8: light chain variable region

QSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWK-NGLMLHQLYPSSH

As used herein, the term "antibody" refers to an anti-HLA-DP antibody that specifically binds to HLA-DP. The scope of the present invention includes antigen-binding fragments of the antibody molecule as well as complete antibody forms that specifically bind to HLA-DP.

A complete antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to the heavy chain by disulfide bonds. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and subclasses include gamma 1 (γ1), gamma 2 (γ2), and gamma 3 (γ3). ), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.

An antigen-binding fragment of an antibody or antibody fragment refers to a fragment having an antigen-binding function, and includes Fab, F(ab'), F(ab')2, Fv, and the like. Among antibody fragments, Fab has a structure having variable regions of light and heavy chains, constant regions of light chains, and a first constant region (CH1) of heavy chains, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. An F(ab')2 antibody is produced by forming a disulfide bond between cysteine residues in the hinge region of Fab'. Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region. Double-chain Fv (two-chain Fv) has a heavy chain variable region and light chain variable region connected by a non-covalent bond, and single-chain Fv (scFv) is generally a heavy chain variable region and a light chain variable region through a peptide linker. They are covalently linked or directly linked at the C-terminus, so that they can form a dimer-like structure like double-chain Fv. Such antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of whole antibodies with papain, and F(ab')2 fragments can be obtained by digestion with pepsin), and gene It can also be produced through recombinant technology.

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

As used herein, the term "heavy chain" refers to a full-length heavy chain comprising a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence for imparting specificity to an antigen and three constant region domains CH1, CH2 and CH3. and fragments thereof. In addition, as used herein, the term "light chain" refers to a full-length light chain and fragments thereof comprising a variable region domain VL and a constant region domain CL comprising an amino acid sequence having a sufficient variable region sequence to impart specificity to an antigen. I mean everything.

Antibodies of the present invention include monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fvs (scFV), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFV) and anti-idiotypic (anti-Id) antibodies, or epitope-binding fragments of such antibodies; and the like, but are not limited thereto.

The monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the same thing except for possible naturally occurring mutations that may be present in minor amounts in the individual antibodies comprising the population. Monoclonal antibodies are highly specific, directed against a single antigenic site. In contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

"Epitope" means a protein determinant to which an antibody can specifically bind. An epitope usually consists of a group of chemically active surface molecules, such as amino acids or sugar side chains, and usually has specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-stereotypic epitopes are distinguished by loss of binding to the former but not to the latter in the presence of a denaturing solvent.

A “humanized” form of a non-human antibody is a chimeric antibody that contains minimal sequence derived from a non-human (eg, murine) immunoglobulin. In most cases, humanized antibodies are derived from a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human, which retains the desired specificity, affinity, and capacity for residues from a hypervariable region of the recipient. It is a human immunoglobulin (recipient antibody) in which residues from a primate hypervariable region have been replaced.

"Human antibody" is a molecule derived from human immunoglobulin, and means that all amino acid sequences constituting the antibody, including complementarity determining regions and structural regions, are composed of human immunoglobulin.

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

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

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

A "framework region" (FR) is a variable domain residue other than a CDR residue. Each variable domain typically has four FRs identified as FR1, FR2, FR3 and FR4.

HLA-DP antibodies are monovalent or bivalent and contain single or double chains. Functionally, the binding affinity of HLA-DP antibodies is in the range of 10 ⁻⁵ M to 10 ⁻¹² M. For example, the binding affinity of the HLA-DP antibody is 10 ^-6 M to 10 ^-12 M, 10 ^-7 M to 10 ^-12 M, 10 ^-8 M to 10 ^-12 M, 10 ^-9 M to 10 ^-12 M M, 10 ^-10 M to 10 ^-12 M, 10 ^-11 M to 10 ^-12 M, 10 ^-5 M to 10 ^-11 M, 10 ^-6 M to 10 ^-11 M, 10 ^-7 M to 10 ^-11 M, 10 ^-8 M to 10 ^-11 M, 10 ^-9 M to 10 ^-11 M, 10 ^-10 M to 10 ^-11 M, 10 ^-5 M to 10 ^-10 M, 10 ^-6 M to 10 ^-10 M, 10 ^-7 M to 10 ^-10 M, 10 ^-8 M to 10 ^-10 M, 10 ^-9 M to 10 ^-10 M, 10 ^-5 M to 10 ^-9 M, 10 ^-6 M to 10 ^-9 M, 10 ^-7 M to 10 ^-9 M, 10 ^-8 M to 10 ^-9 M, 10 ^-5 M to 10 ^-8 M, 10 ^-6 M to 10 ^-8 M, 10 ^-7 M to 10 ^-8 M, 10 ^-5 M to 10 ^-7 M, 10 ^-6 M to 10 ^-7 M or 10 ^-5 M to 10 ^-6 M.

On the other hand, the definition of the CDR sequence (region) may be slightly different even for antibodies having the same variable region according to the numbering scheme, which will be easily understood by those skilled in the art.

The antibody or antibody fragment of the present invention may include not only the sequence of the anti-HLA-DP antibody of the present invention described herein, but also a biological equivalent thereof to the extent that it can specifically recognize HLA-DP. For example, additional changes may be made to the amino acid sequence of the antibody to further improve its binding affinity and/or other biological properties. Such modifications include, for example, deletions, insertions and/or substitutions of residues in the amino acid sequence of the antibody. Such amino acid variations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape and type of amino acid side chain substituents revealed that arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Accordingly, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

Considering the mutations having the above-described biologically equivalent activity, the antibodies of the present invention or the nucleic acid molecules encoding them are construed to include sequences showing substantial identity with the sequences described in SEQ ID NOs. The above substantial identity is at least 90% when the sequence of the present invention and any other sequence described above are aligned so as to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. It refers to sequences exhibiting homology, most preferably at least 95% homology, 96% or more, 97% or more, 98% or more, 99% or more homology.

For example, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 comprising a sequence having 90% or more homology to the sequence of SEQ ID NO: 1 and a sequence having 90% or more homology to the sequence of SEQ ID NO: 2 as another aspect. a heavy chain variable region comprising heavy chain CDR2 and heavy chain CDR 3 comprising a sequence having 90% or more homology with the sequence of SEQ ID NO: 3; And / or a light chain CDR1 comprising a sequence having 90% or more homology to the sequence of SEQ ID NO: 4, a light chain CDR2 comprising a sequence having 90% or more homology to the sequence of SEQ ID NO: 5, and the sequence of SEQ ID NO: 6 It may be an antibody or an antigen-binding fragment thereof that specifically binds to HLA-DP, including a light chain variable region including light chain CDR 3 including sequences having 90% or more homology.

In the present invention, the antibody or antigen-binding fragment thereof may be an antibody or antigen-binding fragment thereof comprising a sequence having at least 90% sequence homology with the heavy chain variable region of SEQ ID NO: 7 in some embodiments.

In the present invention, the antibody or antigen-binding fragment thereof may be an antibody or antigen-binding fragment thereof comprising a sequence having at least 90% sequence homology with the light chain variable region of SEQ ID NO: 8 in some embodiments.

Alignment methods for sequence comparison are known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NBCI, etc., and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the Internet. The BLAST is accessible at www.ncbi.nlm.nih.gov/BLAST/. A sequence homology comparison method using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

Based thereon, an antibody or antigen-binding fragment thereof of the present invention may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the specified sequence described herein or as a whole. , 99%, or greater homology. Such homology can be determined by sequence comparison and/or alignment by methods known in the art. For example, a sequence comparison algorithm (ie, BLAST or BLAST 2.0), manual alignment, or visual inspection can be used to determine the percent sequence homology of a nucleic acid or protein of the invention.

In another aspect, the present invention relates to a nucleic acid encoding the antibody or antigen-binding fragment thereof.

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

"Nucleic acid" has the meaning of comprehensively including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are basic structural units in nucleic acids, include not only natural nucleotides, but also analogs in which sugar or base sites are modified. . The sequences of nucleic acids encoding the heavy and light chain variable regions of the present invention may be modified. Such modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

DNA encoding the antibody is readily isolated or synthesized using conventional procedures (eg, by using oligonucleotide probes capable of specifically binding to DNA encoding the heavy and light chains of the antibody). Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

As used herein, the term "vector" refers to a plasmid vector as a means for expressing a gene of interest in a host cell; cosmid vector; and viral vectors such as bacteriophage vectors, adenoviral vectors, retroviral vectors, and adeno-associated viral vectors. In the vector, the nucleic acid encoding the antibody is operably linked to a promoter.

"Operably linked" means a functional linkage between a nucleic acid expression control sequence (eg, a promoter, signal sequence, or array of transcriptional regulator binding sites) and another nucleic acid sequence, whereby the control sequence is linked to the other nucleic acid. It will regulate the transcription and/or translation of the sequence.

When prokaryotic cells are used as hosts, a strong promoter capable of promoting transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), a ribosome binding site for initiation of translation, and a transcription/translation termination sequence. Further, for example, when a eukaryotic cell is used as a host, a promoter derived from the genome of a mammalian cell (eg, a metallotionine promoter, a β-actin promoter, a human hemoglobin promoter, and a human muscle creatine promoter) or a mammal Promoters derived from viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, Promoters of Moloney Virus (Epstein Barr Virus (EBV) and Rouss Sarcoma Virus (RSV)) can be used, and usually have a polyadenylation sequence as a transcription termination sequence.

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

The vector contains an antibiotic resistance gene commonly used in the art as a selectable marker, for example, for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline. There is a resistance gene.

In another aspect, the present invention relates to a cell transformed with the nucleic acid or the above-mentioned vector. Cells used to generate the antibodies of the present invention may be, but are not limited to, prokaryotic, yeast or higher eukaryotic cells.

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

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

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

The cells can be cultured in various media. Among commercially available media, it can be used as a culture medium without limitation. All other necessary supplements known to those skilled in the art may also be included in suitable concentrations. Culture conditions such as temperature, pH, etc. are already in use with host cells selected for expression and will be apparent to those skilled in the art.

The antibody or antigen-binding fragment thereof may be recovered by, for example, centrifugation or ultrafiltration to remove impurities, and the resultant may be purified using, for example, affinity chromatography. Additional other purification techniques may be used, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, and the like.

In the present invention, the antibody or antigen-binding fragment thereof can be used to study the pathogenesis of immune diseases.

In some embodiments, the antibody or antigen-binding fragment thereof according to the present invention can be used in various in vitro and in vivo experiments using the antibody to study the pathogenesis of immune diseases. For example, the antibody can be used for experiments such as immunoprecipitation (IP), Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), and fluorescence-activated cell sorting (FACS). In particular, the present invention has the advantage that it can be used in flow cytometry, unlike the commercially available HLA-DPA1 antibody (NB-A3), and can be produced in large quantities and can be used by additionally conjugating fluorescence.

In the present invention, the immune disease can be used to study autoimmune diseases and transplant-related diseases, such as diabetes, rheumatoid arthritis, systemic lupus erythematosus, sarcoidosis, liver transplantation, etc., and viral hepatitis, such as chronic hepatitis B or chronic hepatitis C It can be used to study hepatitis, and can be used to study leukemia and cervical cancer as neoplastic diseases, but is not limited thereto.

In some embodiments, the immune disease is Graves' disease, chronic beryllium disease, juvenile chronic arthritis, sarcoidosis, atopic myelitis, polyangiitis ) and granulomatosis, but is not limited thereto.

The present invention may be provided in the form of an antibody-drug conjugate in which a drug is conjugated to the anti-HLA-DP antibody or antigen-binding fragment thereof.

The anti-HLA-DP antibody or antigen-binding fragment thereof may be coupled to a drug through a linker. The linker is a linking site between an anti-HLA-DP antibody or an antigen-binding fragment thereof and a drug. For example, the linker is cleavable in an intracellular condition, that is, in an intracellular environment, the linker is cleaved by the drug in the antibody. to be released through

The linker may be cleaved by a cleavage agent present in the intracellular environment, for example, lysosomes or endosomes, for example, a peptide linker that may be cleaved by an intracellular peptidase or protease enzyme, for example, a lysosomal or endosomal protease. can be Generally, the peptide linker has a length of at least 2 or more amino acids. The cleavage agent may include cathepsin B, cathepsin D, and plasmin, and hydrolyzes the peptide to release the drug into target cells.

The peptide linker can be cleaved by the thiol-dependent protease cathepsin-B, which is overexpressed in cancer tissues, and for example a Phe-Leu or Gly-Phe-Leu-Gly linker can be used. In addition, the peptide linker can be cleaved by, for example, an intracellular protease, and may be a Val-Cit linker or a Phe-Lys linker.

In one embodiment, the cleavable linker is pH sensitive and can be sensitive to hydrolysis at a specific pH value. In general, pH sensitive linkers indicate that they can be hydrolyzed under acidic conditions. For example, acidic labile linkers that can be hydrolyzed in lysosomes, such as hydrazones, semicarbazones, thiosemicarbazones, cis_aconitic amides, orthoesters, acetals, ketals, etc. can

In another aspect, the linker may be cleaved under reducing conditions, for example, a disulfide linker may correspond thereto. SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl -alpha-methyl_alpha-(2-pyridyl-dithio)toluene) can form various disulfide bonds.

In addition, the linker may be, for example, a non-cleavable linker, and the drug is released through only one step of antibody hydrolysis to produce, for example, an amino acid-linker-drug complex. This type of linker can be a thioether group or a maleimidocaproyl group, and can maintain stability in blood.

The drug in the antibody-drug conjugate may be bound to the antibody as an agent that exhibits pharmacological effects, and specifically may be a chemotherapeutic agent, toxin, microRNA (miRNA), siRNA, shRNA, or radioactive isotope. The chemotherapeutic agent may be, for example, a cytotoxic agent or an immunosuppressive agent. Specifically, it may include microtubulin inhibitors, mitotic inhibitors, topoisomerase inhibitors, or chemotherapeutic agents that can function as DNA intercalators. It may also include immunomodulatory compounds, anticancer agents and antiviral agents, or combinations thereof.

The present invention may also be provided in the form of a bispecific antibody in which the anti-HLA-DP antibody or antigen-binding fragment thereof is coupled with an antibody that binds to another antigen.

An anti-HLA-DP antibody or an antigen-binding fragment thereof according to the present invention and an antibody that forms a double antibody can be used without limitation, but an antibody that can specifically bind to an antigen associated with an immune disease is preferred.

Meanwhile, in the present invention, in the process of selecting a hybridoma for producing the monoclonal antibody, a hybridoma for producing another monoclonal antibody capable of producing an additional antibody having a level of specificity and sensitivity similar to that of the antibody was identified. .

Accordingly, in another aspect, the present invention provides a heavy chain variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3; And / or a light chain variable region comprising CDR1 of SEQ ID NO: 9, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 10; It relates to an antibody or antigen-binding fragment thereof that specifically binds to HLA-DP, including.

SEQ ID NO: 1: heavy chain CDR1

GFTFSSYG

SEQ ID NO: 2: heavy chain CDR2

ISRGDSYT

SEQ ID NO: 3: heavy chain CDR3

ARTFAY

SEQ ID NO: 9: light chain CDR1

QSLLDSDGKTY

SEQ ID NO: 5: light chain CDR2

LVS

SEQ ID NO: 10: light chain CDR3

WQGTHFPQT

In the present invention, the antibody or antigen-binding fragment thereof may be an antibody or antigen-binding fragment thereof comprising the heavy chain variable region of SEQ ID NO: 7, but is not limited thereto.

SEQ ID NO: 7: heavy chain variable region

VQLQESGGDLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLEWVATISRGDSYTYYPDSVKGRFAISRDNAKNTLYLQMSSLKSEDTATYYCARTFAYWGQGTLVTVSA

In the present invention, the antibody or antigen-binding fragment thereof may be an antibody or antigen-binding fragment thereof, including the light chain variable region of SEQ ID NO: 11, but is not limited thereto.

SEQ ID NO: 11: light chain variable region

VMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGT

In another aspect, the antibody or antigen-binding fragment thereof is a heavy chain CDR1 comprising a sequence having 90% or more homology to the sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising a sequence having 90% or more homology to the sequence of SEQ ID NO: 2 and a heavy chain variable region comprising a heavy chain CDR 3 comprising a sequence having 90% or more homology with the sequence of SEQ ID NO: 3; And / or a light chain CDR1 comprising a sequence having 90% or more homology with the sequence of SEQ ID NO: 9, a light chain CDR2 comprising a sequence having 90% or more homology with the sequence of SEQ ID NO: 5, and a sequence of SEQ ID NO: 10 It may be an antibody or an antigen-binding fragment thereof that specifically binds to HLA-DP, including a light chain variable region including light chain CDR 3 including sequences having 90% or more homology.

In the present invention, the antibody or antigen-binding fragment thereof may be an antibody or antigen-binding fragment thereof comprising a sequence having at least 90% sequence homology with the heavy chain variable region of SEQ ID NO: 7 in some embodiments.

In the present invention, the antibody or antigen-binding fragment thereof may be an antibody or antigen-binding fragment thereof comprising a sequence having at least 90% sequence homology with the light chain variable region of SEQ ID NO: 11 in some embodiments.

Example

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

Example 1. Experimental method

1-1. cells and reagents

293T cells (CRL-3216) and THP-1 cells (TIB-202) were obtained from ATCC (American Type Culture Collection, Rockville, MD, USA). Polyethylenimine (PEI) is manufactured by Polyscience Inc. (Niles, IL, USA). PE-Cy™7 mouse anti-human HLA-DR antibody (cat.no.560651), PE mouse anti-human HLA-DP antibody (cat.no.566825), FITC goat anti-mouse Ig antibody (cat.no. 554001) and PE goat anti-mouse Ig (cat.no.550589) were purchased from BD Biosciences (San Jose, CA, USA). PE/Cyanine7 anti-human HLA-DR, DP, DQ (cat.no.361708) and PE anti-human HLA-DQ (cat.no.318105) were purchased from Biolegend (San Diego, CA, USA). Purified HLA-DPA1 antibody (NB-A3) was from Santa Cruz Biotec. Inc (CA, USA).

1-2. Immunization and hybridoma cell production

To use HLA-DPA1 as an immunogen, human HLA-DPA1 nucleotide sequence information was obtained from the NCBI genetic database (NM_001242524.1). The length of the human HLA-DPA1 base sequence is 777 bp, and a 789 bp base sequence was synthesized through codon optimization. The synthesized base sequence shows a sequence of 263 amino acids (FIG. 3). The synthesized HLA-DPA1 nucleotide sequence was cloned and inserted into the pET23b vector. For convenience of purification, a vector with a 6-His tag at the c-terminus was used. The cloned vectors were transformed into four strains of E. coli. In order to confirm that HLA-DPA1 is expressed in the transformed cells, Western blotting and affinity binding tests were performed, and it was confirmed that HLA-DPA1 was expressed at about 34 kDa. His-tagged HLA-DPA1 protein was obtained using the Ni-NTA affinity binding test.

The synthesized HLA-DPA1 was mixed with complete freund's adjuvant and injected into female BALB/c mice. After 2 weeks, serum was collected from mouse tails and antibody titers were measured using ELISA. The immunogen HLA-DPA1 was mixed with PBS and injected back into the mice. After 3 days, the spleen was removed from the mouse, washed with a culture medium, and then the spleen cells were isolated. Isolated spleen cells were fused with myeloma cells (Sp2/0-Ag14) using PEG. The fused cells were cultured in 1 x HAT culture medium. Production of the HLA DPA1 gene construct and production of the HLA-DPA1 hybridoma clone was performed by Youngin Frontier Inc. (Seoul, Korea).

1-3. Enzyme-Linked Immunosorbent Assay (ELISA)

HLA-DRA1 (Cloud-Clone Corp., Wuhan, China), HLA-DPA1 (Youngin Frontier Inc. Seoul, Korea) and HLA-DQA1 (Novus Biologicals, USA) proteins were diluted in PBS (100 ng/well) and 96 - Put into a well plate and incubate overnight at 4 ℃. After washing the plate once with PBS, 100ul of blocking buffer was added and incubated at 37 °C for 1 hour. After washing three times with PBS, 100ul of the hybridoma cell supernatant was added and incubated at 37 °C for 1 hour. After washing again with PBS three times, HRP-coupled goat anti-mouse IgG was added and incubated at 37° C. for 1 hour. After washing three times with PBS, 50 ul/well of TMB reacting to HRP was added, reacted at room temperature to develop sufficient color, and after stopping the reaction with 1N H ₂ SO ₄ , the absorbance was checked at a wavelength of 450 nm to confirm binding affinity. .

1-4. flow cytometry

Flow cytometry was performed to select only hybridoma clones producing antibodies capable of specifically binding to the tertiary structure of HLA-DP among the hybridoma cells selected in the ELISA screening. First, THP-1 cells were harvested and added to FACS tubes with 1 x 10 ⁵ cells per tube. Then, 100ul of each hybridoma cell supernatant was added and incubated at 4°C for 30 minutes. After that, 1 ml of FACS buffer was added and centrifuged at 13000 rpm for 5 minutes. The precipitated cell pellet was resuspended in FACS buffer containing FITC goat anti-mouse Ig antibody and incubated at 4°C for 30 minutes. After washing under the same conditions, the pellet was resuspended in 200ul of FACS buffer and analyzed using a FACScalibur (Becton Dickinson, USA). Flow cytometry was performed to screen HLA-DR, HLA-DP and HLA-DQ transfected cells for HLA-DP MAb. Cells were harvested and centrifuged at 13000 rpm for 5 minutes. An antibody mixture containing HLA-DR, HLA-DP, HLA-DQ and HLA class II antibodies was added and incubated at 4 °C for 30 min. After washing with FACS buffer in the same manner, fluorescently labeled cells were analyzed with LSR II (BD Biosciences, San Jose, CA, USA).

1-5. Limiting dilution assay (LD)

A marginal dilution analysis was performed to select a polyclonal as a monoclonal. Briefly, polyclones that bind to HLA-DP were cultured in 100 mm culture dishes and harvested using a serological pipette. Centrifuged at 1300 rpm for 5 minutes and counted using a hematocytometer. One cell per well in a 96-well culture plate was plated with 1 x Hybridoma Fusion and Cloning Supplement (HFCS; Roche, Basel, Switzerland), 1% Penicillin-Streptomycin (P/S; Gibco, USA) and 20% heat inactivation. were seeded in Dulbecco's Modified Eagle's Medium (DMEM; Biowest, Nuaille, France) containing fermented fetal bovine serum (FBS; Biowest, Nuaille, France). Clones with high affinity were selected and seeded in 24 well culture plates. After 2 days, flow cytometry was performed in the same manner. Selected clones were transferred to 6 well culture plates and seeded. Finally, after 2 days flow cytometry was performed and two clones were selected. Selected clones were seeded in 100 mm culture dishes. The entire process was repeated 3-4 times to obtain hybridoma clones producing complete monoclonal antibodies.

1-6. monoclonal antibody purification

In order to obtain a monoclonal antibody from the finally selected clone, it was cultured in a 100 mm culture dish. Hybridoma SFM (Gibco, Waltham, MA, USA) was added every 2 to 3 days to obtain as many antibodies as possible from the same amount of supernatant. When the total volume reached 50 ml, it was harvested and centrifuged at 13000 rpm for 5 minutes, and the supernatant was collected and purified using the Pierce ™ Protein A IgG Purification Kit (Thermo Fisher, Waltham, MA, USA).

1-7. Cloning of HLA-DR, HLA-DP and HLA-DQ

1-7-1. RNA extraction and cDNA synthesis

All samples were randomly collected and human blood from healthy donors was used. mRNA was isolated using the RNeasy®Mini Kit (Qiagen, Hilden, Germany) and cDNA was isolated using AccuPower®CycleScript™ RT PreMix (Bioneer, Korea) and a T100 Thermal Cycler (Bio-rad Laboratories, Hercules, CA, USA). synthesized.

1-7-2. Construction of HLA-DR, HLA-DP and HLA-DQ vectors

First, a pair of primers including Nhe1 and EcoR1 containing the entire open reading frames of HLA-DRA1, HLA-DRB1, HLA-DPA1, HLA-DPB1, HLA-DQA1 and HLA-DQB1 were designed. Each gene was PCR amplified from human cDNA. The sequences of the primers are shown in Table 1. The size of the PCR product was checked by electrophoresis on a 1% agarose gel and purified using a PCR purification kit (Lobopass, Korea). Then, the cDNA fragment was inserted into pGEM®-T easy vector (promega, USA) and transformed into DH5α competent cells (RBC, Taiwan), followed by 50ug/mL ampicillin, 40ug/mL X-gal, and 0.5mM IPTG. Plated on LB plate. After culturing the white colonies on the LB plate overnight in 5mL LB ampicillin (50ug/mL) broth, plasmid DNA was isolated using a plasmid DNA extraction mini kit (Favorgen, Ping-Tung, Taiwan). The extracted plasmid DNA was sequenced through a sequencing service (cosmo-genetech, Korea), and whether the target gene was inserted into the vector was confirmed through electrophoresis, and after treatment with Nhe1 (NEB) and EcoR1 (NEB) restriction enzymes, gel Gel extraction was performed with an extraction kit (Lobopass, Korea), and the product was inserted into a pCDH-EF1-MCS-IRES-copGFP vector and a pcDNA3.1 expression vector (Addgene, Cambridge, MA, USA). Transformation, plasmid extraction, restriction enzyme treatment, electrophoresis, and sequencing were performed in the same manner as the previous procedure.

5’ to 3’ Forward Reverse HLA-DRA ACGGCTAGCGCCACCATGGCCATAAGTGGAGTCCC CGTGAATTCTTACAGAGGCCCCCTGCGTT HLA-DRB1 ACGGCTAGCGCCACCATGGTGTGTCTGAGGCTCCC CGTGAATTCTCAGCTCAGGAATCCTGTTG HLA-DPA1 ACGGCTAGCGCCACCATGCGCCCTGAAGACAGAAT CGTGAATTCTCACAGGGTCCCCTGGGCCC HLA-DPB1 ACGGCTAGCGCCACCATGATGGTTCTGCAGGTTTC ACGCTCGAGATGATGGTTCTGCAGGTTTC HLA-DQA1 ACGGCTAGCGCCACCATGATCCTAAACAAAGCTCT CGTGAATTCTCACAATGGCCCTTGGTGTC HLA-DQB1 ACGGCTAGCGCCACCATGTCTTGGAAGAAGGCTTT CGTGAATTCTCAGTGCAGAAGCCCTGCTG

1-7-3. Transient Transfection

The day before transfection, HEK293T cells were seeded into 6-wells at 5×10 ⁵ per well. Right before transfection, the medium was replaced with 10% DMEM without antibiotics. Cells were transfected with pCDH-DRA plasmid and pCDH-DRB1 using polyethyleneimine (PEI) transfection reagent (same procedure for DP and DQ). The concentration ratio of PEI and plasmid was 5:1. After 6 hours, the medium was changed to 10% DMEM without antibiotics, and 48 hours after transfection, GFP expression was confirmed and FACS analysis was performed.

1-8. Isotyping of anti-HLA-DPA1 monoclonal antibodies

Isotyping was performed to confirm the heavy and light chain types of the resulting anti-HLA-DPA1 monoclonal antibodies. The culture medium obtained by culturing the hybridomas was isotyped using the Rapid ELISA Mouse mAb Isotyping Kit (Thermo Fisher, Waltham, MA, USA) according to the manufacturer's instructions.

1-9. Heavy and light chain variable region sequencing of anti-HLA DPA1 monoclonal antibodies

Sequencing of the heavy and light chain variable domains of the monoclonal antibody was performed to confirm the nucleotide sequence (or amino acid sequence) of the hybridoma cell line produced. Primers used are listed in Table 2 and Table 3. After culturing the hybridoma cell line, total RNA was extracted using RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. DNA was synthesized using AccuPower cyclescript RT premix (Bioneer, Korea) according to the manufacturer's instructions. First PCR was performed using the primers in Tables 2 and 3 to confirm the sequences of the heavy and light chain variable domains of the HLA DPA1-specific antibody. The first PCR result was diluted with DW and the second PCR was performed. Secondary PCR results were purified using a PCR purification kit (Lobopass, Korea) according to the manufacturer's instructions, and sequencing services were provided by Cosmogenetech.

PCR Primer Sequences for Hybridoma Heavy Chain Sequencing 5’ to 3’ Forward Reverse 5′ MSVHE GGG AAT TCG AGG TGC AGC TGC AGG AGT CTG G 3′ Cγ1 outer GGA AGG TGT GCA CAC CGC TGG AC 3′ Cγ2b outer GGA AGG TGT GCA CAC TGC TGG AC 3′ Cγ2a outer GGA AGG TGT GCA CAC CAC TGG AC 3′ Cγ1 inner GCT CAG GGA AAT AGC CCT TGA C 3′ Cγ2a inner GCT CAG GGA AAT AAC CCT TGA C 3′ Cγ2b inner ACT CAG GGA AGT AGC CCT TGA C

PCR Primer Sequences for Hybridoma Light Chain Sequencing 5' to 3' Forward Reverse 5′L-Vκ_3 TGC TGC TGC TCT GGG TTC CAG 3′mCκ GAT GGT GGG AAG ATG GAT ACA GTT 5′L-Vκ_4 ATT WTC AGC TTC CTG CTA ATC 5′L-Vκ_5 TTT TGC TTT TCT GGA TTY CAG 5′L-Vκ_6 TCG TGT TKC TST GGT TGT CTG 5′L-Vκ_6,8,9 ATG GAA TCA CAG RCY CWG GT 5′L-Vκ_14 TCT TGT TGC TCT GGT TYC CAG 5′L-Vκ_19 CAG TTC CTG GGG CTC TTG TTG TTC 5′L-Vκ_20 CTC ACT AGC TCT TCT CCT C 5' mV kappa GAY ATT GTG MTS ACM CAR WCT MCA

Example 2. Development of polyclonal hybridomas for HLA-DPA1

HLA-DPA1 protein was used as an immunogen to produce antibodies specifically reactive to HLA-DP. First, the HLA-DPA1 gene was synthesized as shown in FIG. 3 to produce hybridomas producing HLA DP-specific antibodies. Translation of the HLA-DPA1 protein, immunization, and production of hybridoma cells were performed by Young In Frontier Co., Ltd. as shown in FIG. 2 . Supernatants of 480 hybridoma cell clones were collected and ELISA was performed using the HLA-DPA1 protein as an antigen. Among the positive clones, the top 23 clones were selected (FIG. 4).

Example 3. Confirmation of specificity of hybridoma cells to HLA-DPA1

3-1. Hybridoma screening by ELISA

To examine the specificity of the produced hybridomas, ELISA was performed using HLA class II α chain antigens (HLA-DRA1, HLA-DPA1, HLA-DQA1) (FIG. 5). All 23 clones responded strongly to HLA-DPA1 (OD, 0.38-1.79) (FIG. 5B), but not to HLA-DRA1 and HLA-DQA1 (OD: less than 0.50) (FIGS. 5A, 5C). As a result, it was confirmed that 23 clones responded specifically to HLA-DPA1.

3-2. Hybridoma screening by flow cytometry

Since the ELISA method selects an antibody that recognizes a linear protein, flow cytometry was performed to select an antibody that recognizes a three-dimensional protein structure so as to effectively recognize an antigen in an in vivo environment. THP-1, a cell line expressing all human HLA on the cell surface (including both class I and class II), was used as an antigen. Of the previously selected 23 clones, only 6 clones #10, #13, #15, #18, #22, #23 were confirmed positive by flow cytometry (FIG. 6).

3-3. Selection of specific monoclonal hybridoma cells using limiting dilution method

Since it is difficult to continuously maintain polyclonal hybridoma cells to produce antibodies with high affinity, limiting dilution (Limiting dilution, LD) was performed. As already confirmed, 23 clones were selected through ELISA, and only 6 clones showed positive results in flow cytometry. Figure 7 is a schematic representation of the selection process. Limiting dilution was performed for the top 6 clones. Of these, 4 clones could not produce stable monoclonal antibodies maintaining high titer during several rounds of limiting dilution, and clones #13 and #23, which maintained high titer during limiting dilution, were selected.

The limiting dilution method for #13 and #23 is as shown in FIG. 7, and the #13 clone went through a total of 4 times of limiting dilution process to finally obtain 13-4-1-26-4 and 13-4-1-26- 14 hybridoma clones were selected (FIG. 8A). As for clone #23, 23-6-46-26 was selected as the final clone in the same manner (FIG. 8b). The selected clones were confirmed to have similar (MFI, 23.9) or higher (MFI, 31.2) affinity compared to the commercially available antibody (MFI, 25.0, positive control) (FIG. 9).

In order to confirm that the results were significant even when quantitatively compared with commercially available antibodies, the supernatant of each hybridoma was collected and purified using an IgG purification kit. Monoclonal hybridoma clone 13-4-1-26-14 (MFI, 537) with the most similar binding affinity to commercial antibody (MFI, 523) when the purified antibody was combined with the same dose (100 ng) as human PBMC ) was selected as the final clone (FIG. 10).

Flow cytometry was performed using the transfected cells (pCDH-DR, pCDH-DP and pCDH-DQ) to confirm that the resulting monoclonal antibodies were indeed HLA-DP specific. As a result, compared to pCDH-DR (MFI, 131) and pCDH-DQ (MFI, 136), pCDH-DP cells (MFI, 352) exhibited higher binding affinity, resulting in final clone 13-4- 1-26-14 It did not respond to HLA-DR and HLA-DQ, but it was confirmed that it responded specifically to HLA-DP.

Example 4. Characterization of anti-HLA-DPA1 monoclonal antibodies

4-1. Isotyping test of anti-HLA-DPA1 monoclonal antibodies by ELISA

An isotyping test was performed to confirm the characteristics of the anti-HLA-DPA1 monoclonal antibody. After purifying the anti-DPA1 monoclonal antibody from the hybridoma supernatant, ELISA was performed using a kit in which immunoglobulin class and subclass antibodies were previously captured. All three monoclonal antibodies belonged to IgG2a heavy chain and kappa light chain (FIG. 12).

4-2. Sequencing of anti-HLA-DP monoclonal antibody variable domains

In order to confirm the sequences of the heavy and light chain variable domains of the resulting anti-HLA-DP monoclonal antibody, cDNA of the hybridoma was extracted. After PCR was performed using the primers in Tables 2 and 3, sequencing analysis was performed. As a result of homology analysis using the NCBI IgBlast program, the identified sequences showed 97.3% homology for the heavy chain and 97.2% for the light chain to mouse germline V, D, and J genes, indicating that these antibodies were homologous in the mouse germline. It was confirmed that the immunoglobulin variable domain was rearranged.

As above, specific parts of the content of the present invention have been described in detail, and for those skilled in the art, these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. It will be clear. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Seoul National University R&DB Foundation <120> Anti-HLA-DP monoclonal antibody and use thereof <130> 1069658 <150> KR 1020210096685 <151> 2021-07-22 <160> 50 <170> KoPatentIn 3.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain CDR1 <400> 1 Gly Phe Thr Phe Ser Ser Tyr Gly 1 5 <210> 2 <211> 8 <212> PRT < 213> Artificial Sequence <220> <223> Heavy Chain CDR2 <400> 2 Ile Ser Arg Gly Asp Ser Tyr Thr 1 5 <210> 3 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain CDR3 <400> 3 Ala Arg Thr Phe Ala Tyr 1 5 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Light Chain CDR1 <400> 4 Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr 1 5 10 <210> 5 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Light Chain CDR2 <400> 5 Leu Val Ser 1 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Light Chain CDR3 <400> 6 Gln His Ile Arg Glu Leu Thr Arg 1 5 <210> 7 <211 > 112 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 7 Val Gln Leu Gln Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly Ser 1 5 10 15 Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly 20 25 30 Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val Ala 35 40 45 Thr Ile Ser Arg Gly Asp Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys 50 55 60 Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Thr Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 110 <210> 8 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 8 Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile 1 5 10 15 Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met 20 25 30 His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu Ile Tyr 35 40 45 Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg Glu Leu Thr Arg Ser 85 90 95 Glu Gly Gly Pro Ser Trp Lys Asn Gly Leu Met Leu His Gln Leu Tyr 100 105 110 Pro Ser Ser His 115 <210> 9 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Light Chain 2_CDR1 <400> 9 Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr 1 5 10 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light Chain 2_CDR3 <400> 10 Trp Gln Gly Thr His Phe Pro Gln Thr 1 5 <210> 11 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> VL_2 <400> 11 Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly Gln Pro 1 5 10 15 Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly 20 25 30 Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser Pro Lys 35 40 45 Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro Asp Arg 50 55 60 Phe Thr Gly Ser Gl y Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg 65 70 75 80 Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly Thr His 85 90 95 Phe Pro Gln Thr Phe Gly Gly Gly Thr 100 105 <210> 12 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer (HLA-DRA) <400> 12 acggctagcg ccaccatggc cataagtgga gtccc 35 <210> 13 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer (HLA-DRA) <400> 13 cgtgaattct tacagaggcc ccctgcgtt 29 <210> 14 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer (HLA -DRB1) <400> 14 acggctagcg ccaccatggt gtgtctgagg ctccc 35 <210> 15 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer (HLA-DRB1) <400> 15 cgtgaattct cagctcagga atcctgttg 29 <210> 16 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer (HLA-DPA1) <400> 16 acggctagcg ccaccatgcg ccctgaagac agaat 35 <210> 17 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer (HLA-DPA1) <400> 17 cgtgaattc t cacagggtcc cctgggccc 29 <210> 18 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer (HLA-DPB1) <400> 18 acggctagcg ccaccatgat ggttctgcag gtttc 35 <210> 19 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer (HLA-DPB1) <400> 19 acgctcgaga tgatggttct gcaggtttc 29 <210> 20 <211> 35 <212> DNA <213> Artificial Sequence <220 > <223> Forward Primer (HLA-DQA1) <400> 20 acggctagcg ccaccatgat cctaaacaaa gctct 35 <210> 21 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer (HLA-DQA1) <400> 21 cgtgaattct cacaatggcc cttggtgtc 29 <210> 22 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer (HLA-DQB1) <400> 22 acggctagcg ccaccatgtc ttggaagaag gcttt 35 <210> 23 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer (HLA-DQB1) <400> 23 cgtgaattct cagtgcagaa gccctgctg 29 <210> 24 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer (MsVHE) <400> 24 gggaattcga ggtgcagctg caggagtctg g 31 < 210> 25 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer (Cgamma1 outer) <400> 25 ggaaggtgtg cacaccgctg gac 23 <210> 26 <211> 23 <212> DNA <213 > Artificial Sequence <220> <223> Reverse Primer (Cgamma2b outer) <400> 26 ggaaggtgtg cacactgctg gac 23 <210> 27 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer (Cgamma2a outer) <400> 27 ggaaggtgtg cacaccactg gac 23 <210> 28 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer (Cgamma1 inner) <400> 28 gctcagggaa atagcccttg ac 22 <210> 29 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer (Cgamma2a inner) <400> 29 gctcagggaa ataacccttg ac 22 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer (Cgamma2b inner) <400> 30 actcagggaa gtagcccttg ac 22 <210> 31 <211> 21 < 212> DNA <213> Artificial Sequence <220> <223> Forward Primer (L-Vkappa_3) <400> 31 tgctgctgct ctgggttcca g 21 <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <220> < 223> Forward Primer (L-Vkappa_4) <400> 32 attwtcagct tcctgctaat c 21 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer (L-Vkappa_5) <400> 33 ttttgctttt ctggattyca g 21 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer (L-Vkappa_6) <400> 34 tcgtgttkct stggttgtct g 21 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer (L-Vkappa_6,8,9) <400> 35 atggaatcac agrcycwggt 20 <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer (L-Vkappa_14) <400> 36 tcttgttgct ctggttycca g 21 <210> 37 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer (L-Vkappa_19) <400> 37 cagttcctgg ggctcttgtt gttc 24 <210> 38 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer (L-Vkappa_20 ) <400> 38 ctcactagct cttctcctc 19 <210> 39 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer (mVkappa) <400> 39 gayattgtgm tsacmcarwc tmca 24 <210> 40 <211 > 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer (mCkappa) <400> 40 gatggtggga agatggatac agtt 24 <210> 41 <211> 777 <212> DNA <213> Homo sapiens <400> 41 cgccctgaag acagaatgtt ccatatcaga gctgtgatct tgagagccct ctccttggct 60 ttcctgctga gtctccgagg agctggggcc atcaaggcgg accatgtgtc aacttatgcc 120 gcgtttgtac agacgcatag accaacaggg gagtttatgt ttgaatttga tgaagatgag 180 atgttctatg tggatctgga caagaaggag accgtctggc atctggagga gtttggccaa 240 gccttttcct ttgaggctca gggcgggctg gctaacattg ctatattgaa caacaacttg 300 aataccttga tccagcgttc caaccacact caggccacca acgatccccc tgaggtgacc 360 gtgtttccca aggagcctgt ggagctgggc cagcccaaca ccctcatctg c cacattgac 420 aagttcttcc caccagtgct caacgtcacg tggctgtgca acggggagct ggtcactgag 480 ggtgtcgctg agagcctctt cctgcccaga acagattaca gcttccacaa gttccattac 540 ctgacctttg tgccctcagc agaggacttc tatgactgca gggtggagca ctggggcttg 600 gaccagccgc tcctcaagca ctgggaggcc caagagccaa tccagatgcc tgagacaacg 660 gagactgtgc tctgtgccct gggcctggtg ctgggcctag tcggcatcat cgtgggcacc 720 gtcctcatca taaagtctct gcgttctggc catgaccccc gggcccaggg gaccctg 777 <210> 42 <211 > 789 <212> DNA <213> Artificial Sequence <220> <223> Codon optimized HLA-DPA1 nucleotide sequence <400> 42 catatgcgcc ctgaagatag gatgttccat ataagagctg ttatcctgag agcactcagc 60 cttgcgttcc tgctgagttt acgaggagct ggtgccataa aagcggatca tgtatcaact 120 tatgccgcgt ttgtacagac tcaccgcccg acaggggagt tcatgtttga atttgatgaa 180 gatgaaatgt tctatgtgga tttagataag aaagagacag tatggcatct ggaagaattt 240 ggccaggcct tttcctttga agctcaaggc gggctggcta acattgctat attgaataac 300 aatttgaata ccttgatcca acgttcgaat catacgcaag cgaccaatga tccgcctgag 360 gtgacctaggtt gag ctgggtcagc cgaacacctt aatttgccac 420 atcgataaat tttttccacc agtgctgaac gtcacgtggc tgtgcaatgg tgaacttgtg 480 actgaaggtg tcgcagaaag tttattcctg ccccgtacag attacagctt tcacaaattt 540 cattacctga cgtttgttcc gtcagcagag gacttctatg actgtcgcgt agaacattgg 600 ggattggacc agccgctcct caaacactgg gaggcccaag agcctattca gatgcctgaa 660 accacggaaa ctgtgctttg tgcactgggt ctggttctgg gcctagttgg cattatcgtc 720 ggcacagttt taattattaa atctttacgt tctggacatg acccccgggc acaggggacc 780 cttctcgag 789 < 210> 43 <211> 263 <212> PRT <213> Homo sapiens <400> 43 His Met Arg Pro Glu Asp Arg Met Phe His Ile Arg Ala Val Ile Leu 1 5 10 15 Arg Ala Leu Ser Leu Ala Phe Leu Leu Ser Leu Arg Gly Ala Gly Ala 20 25 30 Ile Lys Ala Asp His Val Ser Thr Tyr Ala Ala Phe Val Gln Thr His 35 40 45 Arg Pro Thr Gly Glu Phe Met Phe Glu Phe Asp Glu Asp Glu Met Phe 50 55 60 Tyr Val Asp Leu Asp Lys Lys Glu Thr Val Trp His Leu Glu Glu Phe 65 70 75 80 Gly Gln Ala Phe Ser Phe Glu Ala Gln Gly Gly Leu Ala Asn Ile Ala 85 90 95 Ile Leu Asn Asn Asn Leu Asn Thr Leu Ile Gln Arg Ser Asn His Thr 100 105 110 Gln Ala Thr Asn Asp Pro Pro Glu Val Thr Val Phe Pro Lys Glu Pro 115 120 125 Val Glu Leu Gly Gln Pro Asn Thr Leu Ile Cys His Ile Asp Lys Phe 130 135 140 Phe Pro Pro Val Leu Asn Val Thr Trp Leu Cys Asn Gly Glu Leu Val 145 150 155 160 Thr Glu Gly Val Ala Glu Ser Leu Phe Leu Pro Arg Thr Asp Tyr Ser 165 170 175 Phe His Lys Phe His Tyr Leu Thr Phe Val Pro Ser Ala Glu Asp Phe 180 185 190 Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Gln Pro Leu Leu Lys 195 200 205 His Trp Glu Ala Gln Glu Pro Ile Gln Met Pro Glu Thr Thr Glu Thr 210 215 220 Val Leu Cys Ala Leu Gly Leu Val Leu Gly Leu Val Gly Ile Ile Val 225 230 235 240 Gly Thr Val Leu Ile Ile Lys Ser Leu Arg Ser Gly His Asp Pro Arg 245 250 255 Ala Gln Gly Thr Leu Leu Glu 260 <210> 44 <211> 500 <212> DNA <213> Artificial Sequence <220> <223> VH <400> 44 gggaattcgg ggtgcagctg caggagtctg ggggagactt agtgaagcct ggagggtccc 60 tgaaactctc ctgtgcagcc tctggattca ctttcagtag ctatggcatg tcttgggttc 120 gccagactcc agacaagagg ctggagtggg tcgcaaccat tagtcgtggt gatagttaca 180 cctactatcc agacagtgtg aaggggcgat tcgccatctc cagagacaat gccaagaaca 240 ccctgtacct gcaaatgagc agtctgaagt ctgaggacac agccacatat tactgtgcca 300 ggacgtttgc ttactggggc caagggactc tggtcactgt ctctgcagcc aaaacaacag 360 ccccatcggt ctatccactg gcccctgtgt gtggagatac aactggctcc tcggtgactc 420 taggatgcct ggtcaagggt tatttccctg agccagtgac cttgacctgg aactctggat 480 ccctgtccag tggtgtgacc 500 <210> 45 <211> 391 <212> DNA <213> Artificial Sequence <220> <223> VL_1-1 <400> 45 gccaatggat gtatttggaa gaagatggat gcgcagacag tctcctgctt ccttagctgt 60 atctctgggg cagaggccca ccatctcata cagggccagc aaaagtgtca gtacatctgg 120 ctatagttat atgcactgga accaacagaa accaggacag ccacccagac tcctcatcta 180 tcttgtatcc aacctagaat ctggggtccc tgccaggttc agtggcagtg ggtctgggac 240 agacttcacc ctcaacatcc atcctgtgga ggaggaggat gctgcaacct attactgtca 300 gcacattagg gagcttacac gttcggaggg gggaccaagc tggaaataaa acgggctgat 360 gctgcaccaa ctgtatccat cttcccacca t 391 <210> 46 <211> 384 <212> DNA <213 > Artificial Sequence <220> <223> VL_1-2 <400> 46 gcgttgggga tttgtgctga cagtctcctg cttccttagc tgtatctctg gggcagaggg 60 ccaccatctc atacagggcc agcaaaagtg tcagtacatc tggctatagt tatatgcact 120 ggaaccaaca gaaaccagga cagccaccca gactcctcat ctatcttgta tccaacctag 180 aatctggggt ccctgccagg ttcagtggca gtgggtctgg gacagacttc accctcaaca 240 tccatcctgt ggaggaggag gatgctgcaa cctattactg tcagcacatt agggagctta 300 cacgttcgga ggggggacca agctggaaat aaaacgggct gatgctgcac caactgtatc 360 catcttccca caatcggcgg aggg 384 <210> 47 <211> 379 <212> DNA <213> Artificial Sequence <220> <223> VL_1-3 <400> agggt gctg tatctctggg gcagagggcc 60 accatctcat acagggccag caaaagtgtc agtacatctg gctatagtta tatgcactgg 120 aaccaacaga aaccaggaca gccacccaga ctcctcatct atcttgtatc caacctagaa 180 tctggggtcc ctgccaggtt cagtggcagt gggtctggga cagacttcac cctcaacatc 240 catcctgtgg aggaggagga tgctgcaacc tattactgtc agcacattag ggagcttaca 300 cgttcggagg ggggaccaag ctggaaataa aacgggctga tgctgcacca actgtatcca 360 tcatccccac catgaaaaa 379 <210> 48 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> VL_2-1 <400> 48 ttgggaacaa cggaccatga tgtgatgacc cagactccac tcactttgtc ggttaccatt 60 ggacaaccag cctccatctc ttgcaagtca agtcagagcc tcttagatag tgatggaaag 120 acatatttga attggttgtt acagaggcca ggccagtctc caaagcgcct aatctatctg 180 gtgtctaaac tggactctgg agtccctgac aggttcactg gcagtggatc agggacagat 240 ttcacactga aaatcagcag agtggaggct gaggatttgg gagtttatta ttgctggcaa 300 ggtacacatt ttcctcagac gttcggtgga ggcaccttgc tggggatcaa acgaggctgc 360 360 <210> 49 <211> 391 <212> DNA <213> Artificial Sequence <220> <223> VL_2-2 <400> 49 gggtca cggt gagttgtaat gacccagact ccactcactt tgtcggttac cattggacaa 60 ccagcctcca tatcttgcaa gtcaagtcag agcctcttag atagtgatgg aaagacatat 120 ttgaattggt tgttacagag gccaggccag tctccaaagc gcctaatcta tctggtgtct 180 aaactggact ctggagtccc tgacaggttc actggcagtg gatcagggac agatttcaca 240 ctgaaaatca gcagagtgga ggctgaggat ttgggagttt attattgctg gcaaggtaca 300 cattttcctc agacgttcgg tggaggcacc aagctggaaa tcaaacgggc tgatgctgca 360 ccaactgtat ccatcttcac accataaaga g 391 <210> 50 <211> 408 <212> DNA <213> Artificial Sequence <220> <223> VL_2-3 <400> 50 ggggtgtggt caaaagaccg atcgccgtag ttgtgatgac cagactccac tcactttgtc 60 ggttaccatt ggacaaccag cctccatctc ttgcaagtca agtcagagcc tcttagatag 120 tgatggaaag acatatttga attggttgtt acagaggcca ggccagtctc caaagcgcct 180 aatctatctg gtgtctaaac tggactctgg agtccctgac aggttcactg gcagtggatc 240 agggacagat ttcacactga aaatcagcag agtggaggct gaggatttgg gagtttatta 300 ttgctggcaa ggtacacatt ttcctcagac gttcggtgga ggcaccaagc tggaaatcaa 360acctgatcacca atctgatcagtgat 408

Claims (6)

a heavy chain variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3; and/or
A light chain variable region comprising CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 6; containing, an antibody that specifically binds to HLA-DP or an antigen-binding fragment thereof.
According to claim 1, comprising a heavy chain variable region of SEQ ID NO: 7, the antibody or antigen-binding fragment thereof.
According to claim 1, comprising a light chain variable region of SEQ ID NO: 8, the antibody or antigen-binding fragment thereof.
A nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 3.
A cell transformed with the nucleic acid of claim 4 or an expression vector containing the same.
A method for producing an antibody or antigen-binding fragment thereof that specifically binds to HLA-DP comprising the following steps:
(a) culturing the cells of claim 5; and
(b) recovering the antibody or antigen-binding fragment thereof from the cultured cells.

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