KR102647825B1 - Anti-HLA-DP monoclonal antibody and use thereof - Google Patents

Abstract

The present invention relates to an anti-HLA-DP monoclonal antibody that binds to HLA-DP with high affinity and specificity and its use, wherein the antibody binds to HLA-DP with high affinity without cross-reacting with HLA-DQ or HLA-DR. It can be used for a variety of purposes, including in vitro cell-based experiments, including immunoblotting and flow cytometry, and in vivo efficacy experiments.

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 that bind 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 multiple genes and polymorphisms. The human leukocyte antigen (HLA) gene is a gene encoding human MHC and is 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 a form bound to a β-microglobulin chain and include HLA-A, -B, and -C. HLA class II consists of α and β chain combinations and includes HLA-DR, -DP, and -DQ. HLA genes composed in this way have the greatest diversity among human genes, and the HLA haplotype, which is composed of a combination of HLA genes, plays a role in distinguishing self from non-self in immune responses. In the immune response, the HLA complex plays a role in inducing an immune response by presenting antigens to T lymphocytes, the core cells of the immune response. Therefore, in the study of the immune response, the study of the HLA complex is a key research field and is being studied in various ways. However, research on HLA Class II has been focused on HLA-DR, and research on HLA-DP has been rapidly increasing recently.

Meanwhile, 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 (HLA-DP related pseudogenes) that are not actually expressed. 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 approximately 9 amino acids is loaded onto the HLA-DP binding site and presented to antigen presenting cells (APCs). Typically, peptides presented by HLA-DP in HLA class II are exogenous antigens and CD4+ T cells recognize and stimulate the peptide-HLA complex. According to the IMGT/HLA database, as of June 2021, there are 2, 2015, 107, 1106, 143, and 1303 proteins known in DRA, DRB1, DPA1, DPB1, DQA1, and DQB1, respectively. Therefore, HLA-DP has the least polymorphism among the three HLA class II molecules. HLA-DP is genetically linked to a variety of diseases, including autoimmune diseases, hepatitis, myeloid diseases, and organ transplants. Autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, diabetes, Graves' disease, chronic berylliosis, juvenile chronic arthritis, sarcoidosis, and atopic myelitis. ), polyangiitis, granulomatosis, etc. 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 recent studies are being conducted on the relationship between peptides for HLA-DP and disease loading. However, HLA-DP is larger than other HLA class II molecules. It has not been extensively studied. Research on HLA and disease mechanisms requires specific isolation, detection, and purification of HLA-DP molecules, and purifying a large amount of HLA-DP molecules requires a particularly large amount of antibodies. Therefore, the present inventors made diligent efforts to develop an effective antibody capable of specifically detecting HLA-DP, and produced a monoclonal hybridoma cell line producing an antibody specific for HLA-DPA1 according to the present invention. The present invention was completed by confirming that the cell line can produce antibodies with improved specificity compared to 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 provides an antibody capable of specifically binding to HLA-DP and a use thereof.

In order to achieve the above object, the present invention provides a heavy chain variable region comprising CDR1 of SEQ ID NO: 1, CDR 2 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, CDR 2 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 by comprising a heavy chain variable region of SEQ ID NO: 7.

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

The present invention also provides a nucleic acid encoding the antibody or antigen-binding fragment thereof.

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

The present invention also provides 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; and

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

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

Figure 1 is a schematic diagram showing the 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 resulting amino acid sequence.
Figure 4 shows the ELISA results of 24 hybridoma clones showing the highest titer among 480 hybridoma clones. Except for #7, all cases were positive for HLA-DPA1 antigen. P represents commercial HLA-DPA1 antibody (NB-A3) as a positive control, and N represents PBS as a negative control.
Figure 5 shows ELISA results for HLAα chain antigen. P represents commercial HLA-DPA1 antibody (NB-A3) as a positive control, and N represents PBS as a negative control. (A) For the DRA1 antigen, all clones showed binding affinity similar to blank. (B) All clones were confirmed positive for the DPA1 antigen, and the five most effective clones were marked in yellow. (C) All clones were confirmed negative for DQA1 antigen.
Figure 6 shows the results of flow cytometry analysis of 23 hybridoma clones confirmed positive in ELISA. The unstained control group to which no antibody was added is indicated in gray, the negative control group to which only the secondary antibody was added is indicated in black, and the positive control group to which a commercial HLA-DPA1 antibody (NB-A3) was added is indicated in light blue. 17 clones were confirmed as negative and shown in blue, and 6 clones were confirmed as positive and shown in red.
Figure 7 is a schematic diagram of screening monoclonal antibodies.
Figure 8 shows the limiting dilution method of hybridoma 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 the final clones. (b) is the result of limiting dilution of clone #23, and 23-6-46-26 was selected as the final clone.
Figure 9 is a flow cytometric analysis result showing the binding affinity of the final clone. The unstained control group to which no antibody was added is indicated in gray, the negative control group to which only the secondary antibody was added is indicated in black, and the positive control group to which a commercial HLA-DPA1 antibody (NB-A3) was added is indicated in light blue. The top three clones selected as final clones are shown in red, and unselected clones are shown in blue.
Figure 10 shows the results of confirming the binding affinity of the purified antibody to human PBMC to confirm clinical applicability. The unstained control group to which no antibody was added is indicated in gray, the negative control group to which only the secondary antibody was added is indicated in black, and the positive control group to which a commercial HLA-DPA1 antibody (NB-A3) was added is indicated in orange. 13-4-1-26-4 is indicated by a red line, 13-4-1-26-14 is indicated by a green line, and 23-6-46-26 is indicated by a blue line.
Figure 11 shows the results of the binding affinity test of the 13-4-1-26-14 clone, quantitatively comparing the binding affinity of the anti-HLA-DPA1 antibody with the binding affinity of a commercially available antibody using HLA transfected cells prepared. did. Controls without antibodies 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.
Figure 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 (gray at the top). , all light chains were identified as kappa chains (bottom gray).
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 otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, the HLA-DPA1 gene is synthesized and used as an immunogen to produce hybridoma cells capable of producing antibodies against HLA-DP, and the hybridoma cells are capable of producing effective monoclonal antibodies. Cells were selected. The monoclonal antibody produced from the finally selected hybridoma cells bound to HLA-DP with higher binding affinity than commercially available antibodies, and when confirmed using human cell lines expressing HLA-DR, DP, and DQ, HLA -DR and did not bind to HLA-DQ, but were 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.

Therefore, in one aspect, the present invention provides a heavy chain variable region comprising CDR1 of SEQ ID NO: 1, CDR 2 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, CDR 2 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 comprising 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 not only the complete antibody form that specifically binds to HLA-DP, but also antigen-binding fragments of the antibody molecule.

A complete antibody has a structure of two full-length light chains and two full-length heavy chains, with each light chain connected to the heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types and is subclassed as gamma1 (γ1), gamma2 (γ2), and gamma3 (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 an antibody fragment refers to a fragment that possesses an antigen-binding function and includes Fab, F(ab'), F(ab')2, and Fv. Among antibody fragments, Fab has a structure that includes the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region (CH1) of the heavy chain, 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. F(ab')2 antibody is produced when cysteine residues in the hinge region of Fab' form a disulfide bond. Fv is the smallest antibody fragment containing only the heavy chain variable region and light chain variable region. Double-chain Fv (two-chain Fv) is a non-covalent bond that connects the heavy chain variable region and the light chain variable region, and single-chain Fv (single-chain Fv, scFv) generally connects the heavy chain variable region and the light chain variable region through a peptide linker. It can be connected by a covalent bond or directly at the C-terminus to form a dimer-like structure, such as double-chain Fv. These antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of the entire antibody with papain, and F(ab')2 fragment can be obtained by digestion with pepsin), and gene It can also be produced through recombinant technology.

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

As used herein, the term “heavy chain” refers to a full-length heavy chain comprising a variable region domain VH and three constant region domains CH1, CH2, and CH3, including an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen. and fragments thereof. In addition, the term "light chain" as used herein refers to a full-length light chain and fragments thereof comprising a variable region domain VL and a constant region domain CL containing an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen. It all means.

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-idiotype (anti-Id) antibodies, or epitope-binding fragments of these antibodies, etc. are included, but are not limited thereto.

The monoclonal antibodies refer to antibodies obtained from a population of substantially homogeneous antibodies, i.e., identical except for possible naturally occurring mutations that may be present in trace amounts in the individual antibodies comprising the population. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

“Epitope” refers to a protein determinant to which an antibody can specifically bind. Epitopes usually consist of groups of chemically active surface molecules, such as amino acids or sugar side chains, and generally have specific three-dimensional structural features as well as specific charge characteristics. Conformational epitopes and non-steric epitopes are distinguished in that binding to the former but not to the latter is lost in the presence of a denaturing solvent.

“Humanized” forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from non-human (eg, murine) immunoglobulins. In most cases, humanized antibodies are derived from a non-human species (donor antibody) that retains the desired specificity, affinity and ability to transfer residues from the hypervariable region of the recipient, such as mouse, rat, rabbit or non-human. It is a human immunoglobulin (recipient antibody) with residues from the primate hypervariable region replaced.

“Human antibody” is a molecule derived from human immunoglobulin, meaning that all amino acid sequences constituting the antibody, including the complementarity determining region and structural region, are composed of human immunoglobulin.

While portions of the heavy and/or light chain are identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, the remaining chain(s) are derived from another species or belong to another antibody class or subclass. Included are “chimeric” antibodies (immunoglobulins) that are identical 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 comprising the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and 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; i.e., CDR1, CDR2, and CDR3) refer to amino acid residues of an antibody variable domain that are required for antigen binding. Each variable domain typically has three CDR regions, identified as CDR1, CDR2 and CDR3.

“Framework regions” (FR) are variable domain residues other than CDR residues. 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.

Meanwhile, the definition of a CDR sequence (region) may be somewhat different for antibodies having the same variable region depending on the numbering method, and this can 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 its biological equivalent, to the extent that it can specifically recognize HLA-DP. For example, additional changes can be made to the amino acid sequence of the antibody to further improve the binding affinity and/or other biological properties of the antibody. Such modifications include, for example, deletions, insertions and/or substitutions of amino acid sequence residues of the antibody. These amino acid mutations are made based on the relative similarity of amino acid side chain substitutions, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape and type of amino acid side chain substitutions shows 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. Therefore, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan, and tyrosine can be said to be biologically equivalent in function.

Considering the mutations having the above-mentioned biological equivalent activity, the antibody of the present invention or the nucleic acid molecule encoding the same is interpreted to also include a sequence showing substantial identity with the sequence shown in SEQ ID NO. The above-mentioned substantial identity is at least 90% when the sequence of the present invention and any other sequence are aligned to the maximum extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art. It means a sequence showing homology, most preferably at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, and at least 99% homology.

For example, the antibody or antigen-binding fragment thereof, in another embodiment, includes a heavy chain CDR1 containing a sequence having more than 90% homology to the sequence of SEQ ID NO: 1, and a sequence having more than 90% homology to the sequence of SEQ ID NO: 2. A heavy chain variable region comprising heavy chain CDR2 and heavy chain CDR 3, which includes a sequence having more than 90% homology to the sequence of SEQ ID NO: 3; and/or a light chain CDR1 containing a sequence having more than 90% homology to the sequence of SEQ ID NO: 4, a light chain CDR2 containing a sequence having more than 90% 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 and includes a light chain variable region including light chain CDR 3 containing a sequence with more than 90% homology.

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

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

Alignment methods for sequence comparison are known in the art. 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. BLSAT can be accessed at www.ncbi.nlm.nih.gov/BLAST/. The sequence homology comparison method using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

On this basis, the antibody or antigen-binding fragment thereof of the present invention has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% compared to the specified sequence or the entire sequence described in the specification. , may have 99% or more homology. Such homology can be determined by sequence comparison and/or alignment by methods known in the art. For example, sequence comparison algorithms (i.e., 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 produced recombinantly by isolating the nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention. The nucleic acid is isolated and inserted into a replicable vector for further cloning (amplification of DNA) or further expression. Based on this, the present invention relates to a vector containing the above nucleic acid from another perspective.

“Nucleic acid” is meant to comprehensively include DNA (gDNA and cDNA) and RNA molecules, and nucleotides, the basic structural unit of nucleic acids, include not only natural nucleotides but also analogues with modified sugar or base sites. . The sequences of nucleic acids encoding the heavy and light chain variable regions of the present invention may be modified. The modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

The DNA encoding the antibody is easily isolated or synthesized using conventional procedures (for example, by using an oligonucleotide probe that can specifically bind to the 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 means for expressing a gene of interest in a host cell, including a plasmid vector; cosmid vector; Includes viral vectors such as bacteriophage vectors, adenovirus 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 (e.g., 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 regulates the transcription and/or translation of the sequence.

When a prokaryotic cell is used as a host, a strong promoter capable of advancing 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. Additionally, for example, in the case of eukaryotic cells as hosts, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter, β-actin promoter, human heroglobin promoter, and human muscle creatine promoter) or mammalian 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 (the promoter of Epstein Barr virus (EBV) and the promoter of Rouss' Sarcoma virus (RSV)) can be used and generally have a polyadenylation sequence as the transcription termination sequence.

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

The vector contains an antibiotic resistance gene commonly used in the art as a selection 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 cells transformed with the nucleic acid or the above-mentioned vector. The cells used to produce the antibodies of the present invention may be, but are not limited to, prokaryotic, yeast, or higher eukaryotic cells.

Strains of the genus Bacillus such as Escherichia coli , Bacillus subtilis and Bacillus thuringensis, Streptomyces , Pseudomonas (e.g. Pseudomonas putida ), Proteus Prokaryotic host cells such as Proteus mirabilis and Staphylococcus (e.g., 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.

From another aspect, the present invention includes the steps of (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. Any commercially available medium 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 used with host cells selected for expression and will be clear to those skilled in the art.

The antibody or antigen-binding fragment thereof can be recovered by, for example, centrifugation or ultrafiltration to remove impurities, and the resulting product can 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, etc.

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 antibodies to study the pathogenesis of immune diseases. For example, the antibody can be used in experiments such as immunoprecipitation (IP), Western Blotting (WB), ELISA (enzyme-linked immunosorbent assay), and flow cytometry (Fluorescence-activated cell sorting, FACS). In particular, the present invention has the advantage that, unlike the commercially available HLA-DPA1 antibody (NB-A3), it can be used in flow cytometry. In addition, it 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, and liver transplantation, and viral hepatitis such as chronic hepatitis B or chronic hepatitis C. It can be used to study hepatitis, and it can be used to study leukemia and cervical cancer as neoplastic diseases, but is not limited to these.

In some embodiments, the immune disease includes Basedow's disease, chronic berylliosis, juvenile chronic arthritis, sarcoidosis, atopic myelitis, and 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 bound to a drug through a linker. The linker is a site that connects an anti-HLA-DP antibody or an antigen-binding fragment thereof and a drug. For example, the linker is in a form that is cleavable under intracellular conditions, that is, the drug cleaves the linker from the antibody in the intracellular environment. so that it can be released through

The linker may be cleaved by a cleaving agent present in the intracellular environment, such as lysosomes or endosomes, for example, a peptide linker that may be cleaved by an intracellular peptidase or protease enzyme, such as a lysosomal or endosomal protease. It can be. Typically, peptide linkers are at least two amino acids long. The cleavage agent may include cathepsin B, cathepsin D, and plasmin, and hydrolyzes the peptide to release the drug into the target cell.

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. Additionally, 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, meaning that it may be susceptible to hydrolysis at certain pH values. In general, pH sensitive linkers indicate that they can be hydrolyzed under acidic conditions. For example, acid labile linkers that can be hydrolyzed in lysosomes, such as hydrazone, semicarbazone, thiosemicarbazone, cis_aconitic amide, orthoester, acetal, ketal, etc. You can.

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

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

The drug in the antibody-drug conjugate is an agent that exhibits pharmacological effects and may be bound to an antibody, and may specifically be a chemotherapeutic agent, toxin, micro RNA (miRNA), siRNA, shRNA, or radioactive isotope. The chemotherapy agent may be, for example, a cytotoxic agent or an immunosuppressant. Specifically, it may include a microtubulin inhibitor, a mitotic inhibitor, a topoisomerase inhibitor, or a chemotherapeutic agent that can function as a DNA intercalator. 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 and an antibody that binds to another antigen are combined.

Antibodies that form double antibodies with anti-HLA-DP antibodies or antigen-binding fragments thereof according to the present invention can be used without limitation, but antibodies that can specifically bind to antigens related to immune diseases are preferred.

Meanwhile, in the present invention, in the process of selecting hybridomas for producing the monoclonal antibody, a hybridoma for producing another monoclonal antibody capable of producing additional antibodies with similar levels of specificity and sensitivity as the above antibody was identified. .

Therefore, from 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.

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 comprising 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 embodiment, the antibody or antigen-binding fragment thereof includes a heavy chain CDR1 comprising a sequence having more than 90% homology to the sequence of SEQ ID NO: 1, and a heavy chain CDR2 comprising a sequence having more than 90% homology to the sequence of SEQ ID NO: 2. and a heavy chain variable region including heavy chain CDR 3, which includes a sequence having more than 90% homology to the sequence of SEQ ID NO: 3; and/or a light chain CDR1 comprising a sequence having more than 90% homology to the sequence of SEQ ID NO: 9, a light chain CDR2 comprising a sequence having more than 90% homology to 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 and includes a light chain variable region including light chain CDR 3 containing a sequence with more than 90% homology.

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

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

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 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.566825). 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 purchased from Santa Cruz Biotec. Purchased from 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 777bp, and a 789bp base sequence was synthesized through codon optimization. The synthesized base sequence represents a sequence of 263 amino acids (Figure 3). The synthesized HLA-DPA1 base 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 vector was transformed into four E. coli strains. Western blot and affinity binding tests were performed to confirm whether HLA-DPA1 was expressed in the transformed cells, 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 then injected into female BALB/c mice. After 2 weeks, serum was collected from the mouse tail and antibody titer was measured using ELISA. The immunogen HLA-DPA1 was mixed with PBS and reinjected into the mouse. After 3 days, the spleen was separated from the mouse, washed with culture medium, and then the spleen cells were isolated. Isolated spleen cells were fused with myeloma cells (Sp2/0-Ag14) using PEG. Confluent cells were cultured in 1 x HAT culture medium. Production of HLA DPA1 gene construct and HLA-DPA1 hybridoma clone were provided by Youngin Frontier Inc. The study was conducted in 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 incubated at 96 -Put it in a well plate and culture it at 4°C overnight. 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 hybridoma cell supernatant was added and incubated at 37°C for 1 hour. After washing again with PBS three times, HRP-conjugated goat anti-mouse IgG was added and incubated at 37°C for 1 hour. After washing three times with PBS, 50ul/well of TMB, which reacts with HRP, was added and reacted at room temperature to develop sufficient color. The reaction was stopped with 1N H ₂ SO ₄ and the binding affinity was confirmed by checking the absorbance at a wavelength of 450 nm. .

1-4. flow cytometry

Among the hybridoma cells selected in the ELISA screening, flow cytometry was performed to select only hybridoma clones that produced antibodies that could specifically bind to the tertiary structure of HLA-DP. 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. Afterwards, 1ml of FACS buffer was added and centrifuged at 13000rpm 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 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 for 30 min at 4 °C. In the same way, the cells were washed with FACS buffer, and the fluorescently labeled cells were analyzed with LSR II (BD Biosciences, San Jose, CA, USA).

1-5. Limiting dilution assay (LD)

Limiting dilution analysis was performed to select polyclones into monoclones. Briefly, polyclones binding 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. Incubate one cell per well in a 96-well culture plate with 1 x Hybridoma Fusion and Cloning Supplement (HFCS; Roche, Basel, Switzerland), 1% penicillin-streptomycin (P/S; Gibco, USA) and 20% heat inactivation. They were seeded in Dulbecco's Modified Eagle's Medium (DMEM; Biowest, Nuaille, France) containing fetal bovine serum (FBS; Biowest, Nuaille, France). Clones with high affinity were selected and then seeded into 24 well culture plates. Two days later, flow cytometry was performed using the same method. 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

To obtain monoclonal antibodies from the finally selected clones, they were cultured in a 100 mm culture dish. Hybridoma SFM (Gibco, Waltham, MA, USA) was added every 2 to 3 days to obtain as much antibody as possible from the same amount of supernatant. When the total volume reached 50 ml, they were harvested and centrifuged at 13000 rpm for 5 min, 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 used human blood from healthy donors. mRNA was isolated using the RNeasy®Mini Kit (Qiagen, Hilden, Germany) and cDNA was isolated using AccuPower®CycleScript™ RT PreMix (Bioneer, Korea) and T100 Thermal Cycler (Bio-rad Laboratories, Hercules, CA, USA). synthesized.

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

First, we designed a pair of primers including Nhe1 and EcoR1, which cover the entire open reading frames of HLA-DRA1, HLA-DRB1, HLA-DPA1, HLA-DPB1, HLA-DQA1, and HLA-DQB1, and used the corresponding primers to Each gene was PCR amplified from human cDNA. The sequences of the primers are as shown in Table 1. The size of the PCR product was confirmed by electrophoresis on a 1% agarose gel and purified using a PCR purification kit (Lobopass, Korea). Afterwards, the cDNA fragment was inserted into pGEM®-T easy vector (promega, USA), transformed into DH5α competent cells (RBC, Taiwan), and incubated with 50ug/mL ampicillin, 40ug/mL X-gal, and 0.5mM IPTG. It was plated on an LB plate. White colonies on the LB plate were cultured overnight in 5mL LB ampicillin (50ug/mL) liquid medium, and then 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. After restriction enzyme treatment using Nhe1 (NEB) and EcoR1 (NEB), the gel Gel extraction was performed using an extraction kit (Lobopass, Korea), and the product was inserted into the pCDH-EF1-MCS-IRES-copGFP vector and pcDNA3.1 expression vector (Addgene, Cambridge, MA, USA). Transformation, plasmid extraction, restriction enzyme treatment, electrophoresis, and base sequencing were performed in the same manner as the previous process.

5’ to 3’ Forward Reverse HLA-DRA ACGGCTAGCGCCACCACATGCCATAAGTGGAGTCCC CGTGAATTCTTACAGAGGCCCCCTGCGTT HLA-DRB1 ACGGCTAGCGCCACCATGGTGTGTCTGAGGCTCCC CGTGAATTCTCAGCTCAGGAATCCTGTTG HLA-DPA1 ACGGCTAGCGCCACCATGCGCCCTGAAGACAGAAT CGTGAATTCTCACAGGGTCCCCTGGGCCC HLA-DPB1 ACGGCTAGCGCCACCATGATGTGTTTCTGCAGGTTTC ACGCTCGAGATGATGGTTCTGCAGGGTTTC HLA-DQA1 ACGGCTAGCGCCACCATGATCCTAAACAAAGCTCT CGTGAATTCTCACAATGGCCCTTGGTGTC HLA-DQB1 ACGGCTAGCGCCACCATGTCTTGGAAGAAGGCTTT CGTGAATTCTCAGTGCAGAAGCCCTGCTG

1-7-3. Transient transfection

The day before transfection, HEK293T cells were inoculated into 6-wells at 5 x 10 ⁵ per well. Immediately 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 treated to be 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 antibody

Isotyping was performed to confirm the heavy and light chain types of the generated 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 antibody

The base sequence (or amino acid sequence) of the produced hybridoma cell line was confirmed by sequencing the heavy and light chain variable domains of the monoclonal antibody. Primers used are listed in Table 2 and Table 3. After culturing the hybridoma cell line, total RNA was extracted using the 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. To confirm the heavy and light chain variable domain sequences of the HLA DPA1-specific antibody, first PCR was performed using the primers in Tables 2 and 3. The results of the first PCR were diluted with DW and the second PCR was performed. The secondary PCR results were purified using a PCR purification kit (Lobopass, Korea) according to the manufacturer's instructions, and sequencing services were performed 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'mVkappa GAY ATT GTG MTS ACM CAR WCT MCA

Example 2. Development of polyclonal hybridoma for HLA-DPA1

HLA-DPA1 protein was used as an immunogen to produce antibodies that specifically react to HLA-DP. First, the HLA-DPA1 gene was synthesized as shown in Figure 3 to produce a hybridoma that produces an HLA DP-specific antibody. Translation of HLA-DPA1 protein, immunization, and production of hybridoma cells were carried out by Youngin Frontier Co., Ltd. through the process shown in Figure 2. Supernatants of 480 hybridoma cell clones were collected and ELISA was performed using HLA-DPA1 protein as antigen. Among the positive clones, the top 23 clones were selected (Figure 4).

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

3-1. Hybridoma screening by ELISA

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

3-2. Hybridoma screening by flow cytometry

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

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

Polyclonal hybridoma cells are difficult to maintain continuously to produce antibodies with high affinity, so limiting dilution (Limiting dilution) is used to obtain monoclonal hybridoma cells that produce monoclonal antibodies that specifically react to HLA-DPA1. LD) was performed. As already confirmed, 23 clones were selected through ELISA, and only 6 clones showed positive results in flow cytometry. Figure 7 schematically shows the selection process. Limiting dilution was performed on the top six clones. Among these, four clones could not produce stable monoclonal antibodies that maintained high titers during multiple limiting dilutions, so clones #13 and #23, which maintained high titers during the limiting dilution process, were selected.

The limiting dilution method for #13 and #23 is as shown in Figure 7, and the #13 clone went through a total of 4 limiting dilution processes and finally became 13-4-1-26-4 and 13-4-1-26- 14 hybridoma clones were selected (Figure 8a). Clone #23 was selected in the same manner, with 23-6-46-26 being the final clone (Figure 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).

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

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

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

4-1. Isotyping test of anti-HLA-DPA1 monoclonal antibody 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 the IgG2a heavy chain and kappa light chain (Figure 12).

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

To confirm the sequences of the heavy and light chain variable domains of the generated anti-HLA-DP monoclonal antibody, cDNA of the hybridoma was extracted. PCR was performed using the primers in Tables 2 and 3, and then sequencing analysis was performed. As a result of homology analysis using the NCBI IgBlast program, the identified sequences showed 97.3% heavy chain and 97.2% light chain homology to the mouse germline V, D, and J genes, indicating that these antibodies are active in the mouse germline. It was confirmed that it was an immunoglobulin variable domain produced by rearrangement.

As above, specific parts of the content of the present invention have been described in detail, and those skilled in the art will understand that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. It will be obvious. 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 Gly 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 cgtgaattct 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 TGATGTCT GCAGGTTTC 29 <210> 20 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Forward Primer (HLA-DQA1) g 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 ctcctt ggct 60 ttcctgctga gtctccgagg agctggggcc atcaaggcgg accatgtgtc aacttatgcc 120 gcgtttgtac agacgcatag accaacaggg gagtttatgt ttgaatttga tgaagatgag 180 atgttctatg tggatctgga caagaaggag accgtctggc atctggagga gtttggccaa 240 gccttttcct ttgaggctca gggc gggctg gctaacattg ctatattgaa caacaacttg 300 aataccttga tccagcgttc caaccacact caggccacca acgatccccc tgaggtgacc 360 gtgtttccca aggagcctgt ggagctgggc cagcccaaca ccctcatctg ccacattgac 420 aagttcttcc caccagtgct caacg tcacg 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 ctgggcc tag 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 ttg tacagac 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 gtgaccgttt ttccaaagga accggttgag ctgggtcagc cgaacacctt aatttgccac 420 atcgataaat tttttccacc agtgctgaac gtcacgtggc tgtgcaatgg tga acttgtg 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 tctt gggttc 120 gccagactcc agacaagagg ctggagtggg tcgcaaccat tagtcgtggt gatagttaca 180 cctactatcc agacagtgtg aagggggcgat tcgccatctc cagagacaat gccaagaaca 240 ccctgtacct gcaaatgagc agtctgaagt ctgaggacac agccacatat tactgtgcca 300 ggacgtttgc ttactggggc caagggactc tggtcactgt ctctgcagcc aaaacaacag 360 ccccatcggt ctatccactg gcccctgtgt gtggagatac aactggctcc t cggtgactc 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 cagagggcca 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 cagtctcc tg 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 tca gcacatt 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> 47 ggggggatt gtgctgacca gtctcctgct tccttagctg tatctctggg gcagagggcc 60 accatctcat acagggccag caaaagtgtc agtacatctg gctatagtta tatgcactgg 120 aaccaacaga aaccaggaca gccacccaga ct cctcatct 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 t gtgatgacc 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 agt ggaggct 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 gggtcacggt gagttgtaat gacccagact ccactcactt tgtcggttac cattggacaa 60 ccagcctcca tatcttgcaa gtcaagtcag agcctcttag atagtgatgg aaagacatat 120 ttgaattggt tgttacagag gccaggccag t ctccaaagc 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 at cgccgtag 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 aaat cagcag agtggaggct gaggatttgg gagtttatta 300 ttgctggcaa ggtacacatt ttcctcagac gttcggtgga ggcaccaagc tggaaatcaa 360acgggctgat gctgcaccaa ctgtatccat cttcaaaacc atcaacaa 408

Claims (6)

A heavy chain variable region comprising CDR1 of SEQ ID NO: 1, CDR 2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3; and
An antibody or antigen-binding fragment thereof that specifically binds to HLA-DP, comprising a light chain variable region comprising CDR1 of SEQ ID NO: 4, CDR 2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 6.
The antibody or antigen-binding fragment thereof according to claim 1, comprising the heavy chain variable region of SEQ ID NO: 7.
The antibody or antigen-binding fragment thereof according to claim 1, comprising the light chain variable region of SEQ ID NO: 8.
A nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 3.
Cells transformed with the nucleic acid of claim 4 or an expression vector containing the same.
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 paragraph 5; and
(b) recovering the antibody or antigen-binding fragment thereof from the cultured cells.