Antibody, antigen-binding fragment thereof, or peptide, which specifically binds to blood type antigen-specific immunoglobulins

Abstract

The present invention relates to an antibody, an antigen-binding fragment thereof, or a peptide, which specifically binds to blood type antigen-specific immunoglobulins. The antibody, antigen-binding fragment thereof, and peptide according to the present invention have immunoglobulin inhibitory activity for preventing RBC aggregation. Accordingly, it is possible to effectively prevent or treat immune diseases that may occur during blood transfusion or organ transplantation between individuals having nonmatching blood types.

Description

An antibody, antigen-binding fragment or peptide thereof that specifically binds to a blood group antigen-specific immunoglobulin

[0001] The present invention relates to an antibody, an antigen-binding fragment thereof or a peptide that specifically binds to a blood group antigen-specific immunoglobulin.

[0002] The ABO blood type has been widely used to classify blood to determine transfusion compatibility. The ABO blood type classification is based on the types of antigens on red blood cells (RBCs), such as type A and type B antigens. Type A blood contains RBCs carrying type A antigens and IgM specific to type B antigens, while type B blood contains RBCs carrying type B antigens and IgM specific to type A antigens. For example, when IgM specific to type B antigens encounters RBCs carrying type B antigens, it causes RBC agglutination, an immune response. The same occurs with type A blood. Therefore, transfusions between individuals with incompatible blood types are, in principle, difficult to achieve. Notably, ABO antigens are known to be found on the surfaces of various organs, making blood type matching essential for organ transplantation.

[0003] Blood transfusions and organ transplants between individuals with different blood types have limitations due to the aforementioned immune response. However, advancements in medical technology have made it possible to significantly reduce the immune response and enable transplants even in cases of blood type mismatch through so-called desensitization therapy. Desensitization therapy primarily involves plasma exchange, with rituximab administered to suppress the immune response by preventing the generation of antibody-producing B cells (Non-patent Document 1). Typically, rituximab is administered first, followed by plasma exchange.

[0004] The inventors of the present invention completed the present invention by studying antibodies and peptides to reduce immune responses that may occur during organ transplantation between different blood types as described above.

[0005] [Prior Art Literature]

[0006] [Non-patent literature]

[0007] (Non-patent Document 1) Barnett AN, Hadjianastassiou VG, Mamode N. Rituximab in renal transplantation. Transl Int. 2013 Jun;26(6):563-75.

[0008] The problem to be solved in the present invention is to provide an antibody, an antigen-binding fragment thereof, or a peptide that can reduce the immune response of immunoglobulin M (Ig M) that specifically binds to blood type A and B antigens based on the ABO blood type.

[0009] The antibody, or antigen-binding fragment thereof, that specifically binds to a blood group antigen-specific immunoglobulin according to the present invention is technically characterized by comprising a CDR3 selected from the group consisting of an amino acid sequence of SEQ ID NO: 8, a CDR3 comprising an amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10.

[0010] The peptide that specifically binds to a blood group antigen-specific immunoglobulin according to the present invention is technically characterized in that it comprises a peptide selected from the group consisting of a peptide comprising an amino acid sequence of SEQ ID NO: 8, a peptide comprising an amino acid sequence of SEQ ID NO: 9, and a peptide comprising an amino acid sequence of SEQ ID NO: 10.

[0011] The method for producing an antibody, an antigen-binding fragment thereof, or a peptide that specifically binds to a blood group antigen-specific immunoglobulin according to the present invention is characterized by comprising the steps of: (a) culturing a cell transformed with a vector containing a nucleic acid encoding the antibody, an antigen-binding fragment thereof, or a peptide; and (b) recovering the antibody, the antigen-binding fragment thereof, or the peptide from the cultured cell and / or the culture medium thereof.

[0012] The pharmaceutical composition for preventing or treating an immune disease according to the present invention is technically characterized by containing, as an active ingredient, an antibody, an antigen-binding fragment thereof, or a peptide that specifically binds to the blood group antigen-specific immunoglobulin.

[0013] The antibodies, antigen-binding fragments thereof, and peptides of the present invention possess immunoglobulin inhibitory activity that prevents RBC aggregation. This allows for the effective prevention or treatment of immune disorders that may arise during blood transfusions or organ transplants between individuals with incompatible blood types.

[0014] Figure 1 is a schematic diagram showing a method for purifying IgM (a) and an electron microscope image of IgM (b).

[0015] Figure 2 is a schematic diagram showing the mimotope screening process of type A and type B antigens of RBC (a) and the results of binding affinity analysis using dynamic laser scattering (DLS) (b, c).

[0016] Figure 3 is a schematic diagram showing the analysis process of selected Fv antibodies (A28, B6, B22).

[0017] Figure 4 shows the results of the concentration-dependent inhibition of the agglutination reaction of selected Fv antibodies (A28, B6, B22) against anti-sera.

[0018] Figure 5 shows the results of concentration-dependent inhibition of agglutination reaction of selected Fv antibodies (A28, B6, B22) against purified IgM.

[0019] Figure 6 shows the results of the concentration-dependent inhibition of the agglutination reaction of selected peptides (A28, B6, B22) against anti-sera and purified IgM.

[0020] Unless otherwise defined, 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 pertains. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

[0021] The term "antibody" as used herein is the most comprehensive description and encompasses all forms of antibodies known to date. That is, as long as an antibody exhibits antigen-binding activity, it encompasses various antibody structures, including, but not limited to, single antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antigen-binding fragments thereof. The term "antibody" includes conventional four-chain antibodies, single-domain antibodies, and antigen-binding fragments thereof.

[0022] The antibody according to the present invention may be in the form of a single domain antibody (sdAb). Single domain antibodies may include, but are not limited to, heavy chain variable domains from heavy chain-only antibodies (e.g., VHH (variable domain of the heavy chain of a heavy chain antibody) in Camelidae), light chains derived from conventional four-chain antibodies, binding molecules naturally lacking a single domain (e.g., VH or VL), humanized heavy chain-only antibodies, human single domain antibodies produced by transgenic mice or rats expressing human heavy chain segments, and engineered domains and single domain scaffolds other than those derived from antibodies. sdAbs may be derived from any species, including, but not limited to, mouse, rat, human, camel, llama, lamprey, fish, shark, goat, rabbit, and bovine. They may also include naturally occurring single domain antibodies from species other than Camelidae.

[0023] Additionally, single-domain antibodies are derived from naturally occurring single-domain antigen-binding molecules known as light-chain-deficient heavy-chain antibodies. Such single-domain molecules are disclosed, for example, in WO 94 / 04678 and Hamers-Casterman et al., (1993) Nature 363:446-448. The variable domain derived from a heavy chain molecule naturally lacking a light chain is referred to herein as a VHH, distinguishing it from the conventional VH of a four-chain immunoglobulin. Such VHH molecules can be derived from antibodies produced by Camelidae species, such as camels, llamas, vicuñas, dromedaries, alpacas, and guanacos. Species other than Camelidae can produce heavy chain molecules naturally lacking light chains, and such VHHs are within the scope of the present disclosure.

[0024] Additionally, a single domain antibody according to the present invention may be a chimeric antibody. Particular chimeric antibodies are described, for example, in U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In some embodiments, a chimeric antibody may comprise a non-human variable region (e.g., a variable region derived from a camelid species, such as a llama) and a human constant region. Additionally, a chimeric antibody may be humanized. Typically, a non-human antibody is humanized to reduce immunogenicity toward humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. The humanized antibody will optionally also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0025] The term "Fv (variable region fragment)" used herein refers to the antigen-binding region of immunoglobulin G, which is composed of three CDR regions (CDR1, CDR2, and CDR3) and a framework region (FR) between the CDR regions, and is the minimum antibody fragment having only a heavy chain variable region and / or a light chain variable region. Recombinant techniques for producing Fv are described in international patent application WO88 / 10649, etc.

[0026] The term "CDR (complementarity determining region)" as used herein refers to the amino acid residues of an antibody variable domain that are necessary for antigen binding. Each variable domain typically has three CDR regions, identified as CDR1, CDR2, and CDR3. The CDRs of the heavy chain variable region (heacy chain) may also be referred to as HCDRs.

[0027] The term "autodisplay" as used herein refers to a technique for displaying a variant polypeptide as a fusion protein with at least a portion of the coat protein on the surface of an organism, such as a phage or E. coli. The utility of autodisplay lies in its ability to rapidly and efficiently isolate sequences that bind with high affinity to a target antigen from a large library of randomized protein variants. Autodisplay of peptide and protein libraries has been used to screen millions of polypeptides for polypeptides with specific binding properties. In particular, generating a diverse library of antibodies or antigen-binding proteins is crucial for high-affinity antibody isolation. The CDR3 region has been found to be frequently involved in antigen binding. Because the CDR3 region on the heavy chain varies considerably in size, sequence, and structural conformation, it can be utilized to generate diverse libraries.

[0028] The term "nucleic acid" encompasses DNA (gDNA and cDNA) and RNA molecules. Nucleotides, the basic structural units of nucleic acids, include not only natural nucleotides but also analogues with modified sugar or base moieties. 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.

[0029] The amino acid sequence of the antibody or antigen-binding fragment thereof of the present invention or the nucleic acid encoding the same is interpreted to also include a sequence showing substantial identity with the sequence described in the sequence number. The substantial identity means a sequence showing at least 90% homology, most preferably at least 95% homology, 96% or more, 97% or more, 98% or more, or 99% or more homology when the sequence of the present invention and any other sequence are aligned to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0030] Based on this, the antibody or antigen-binding fragment thereof of the present invention may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homology to the specified sequences or the entirety of the sequences described in the specification. Such homology may be determined by sequence comparison and / or alignment using methods known in the art. For example, the percent sequence homology of the nucleic acids or proteins of the present invention may be determined using sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection.

[0031] The DNA encoding the antibody is readily isolated or synthesized using conventional procedures (e.g., by using an oligonucleotide probe that specifically binds to DNA encoding the heavy and light chains of the antibody). Many vectors are available. Vector components typically 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.

[0032] The term "vector" as used herein refers to a means for expressing a target gene in a host cell, including plasmid vectors; cosmid vectors; viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors. In the vector, a nucleic acid encoding an antibody is operably linked to a promoter.

[0033] "Operably linked" means a functional association between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, whereby the regulatory sequence regulates transcription and / or translation of the other nucleic acid sequence.

[0034] In the case of a prokaryotic cell as a host, it is common to include a strong promoter that can drive transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter), a ribosome binding site for translation initiation, and a transcription / translation termination sequence. In addition, for example, when a eukaryotic cell is used as a host, promoters derived from the genome of mammalian cells (e.g., metallothionine promoter, actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian 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, promoter of Moloney virus, promoter of Epstein-Barr virus (EBV), and promoter of Rous sarcoma virus (RSV)) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.

[0035] 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).

[0036] The above vector contains antibiotic resistance genes commonly used in the art as selectable markers, for example, resistance genes for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

[0037] In another aspect, the present invention relates to a cell transformed with the aforementioned vector. The cell used to produce the antibody of the present invention may be, but is not limited to, a prokaryotic cell, a yeast cell, or a higher eukaryotic cell. In the case of prokaryotes, prokaryotic host cells such as Bacillus strains, such as Escherichia coli (hereinafter "E. coli"), Bacillus subtilis, and Bacillus thuringiensis, and Streptomyces, Pseudomonas, Proteus mirabilis, and Staphylococcus may be used.

[0038] In another aspect, the present invention relates to a method for producing an antibody or an antigen-binding fragment thereof, comprising the steps of (a) culturing cells; and (b) recovering the antibody or an antigen-binding fragment thereof from the culture medium and / or the cells.

[0039] The above cells can be cultured in various media. Any commercially available medium can be used as a culture medium. Any other essential supplements known to those skilled in the art may also be included at appropriate concentrations. Culture conditions, such as temperature, pH, etc., are already used with the host cells selected for expression and will be readily apparent to those skilled in the art.

[0040] The term "transformation" as used herein refers to introducing a vector containing a nucleic acid encoding a target protein into a host cell so that the protein encoded by the nucleic acid can be expressed within the host cell. The transformed nucleic acid includes all nucleic acids, whether integrated into the chromosome of the host cell or located extrachromosomally, as long as they can be expressed within the host cell. Furthermore, the nucleic acid includes DNA and RNA encoding the target protein. The nucleic acid may be introduced in any form as long as it can be introduced into the host cell and expressed. For example, the nucleic acid may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for autonomous expression. The expression cassette typically includes a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, all of which are operably linked to the nucleic acid. The expression cassette may be in the form of a self-replicating expression vector. Additionally, the nucleic acid may be introduced into the host cell in its own form and operably linked to a sequence necessary for expression in the host cell.

[0041] The antibody or antigen-binding fragment thereof can be recovered by removing impurities, for example, by centrifugation or ultrafiltration, and the resultant product can be purified, for example, by using affinity chromatography. Additional purification techniques, for example, anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, etc., can be used.

[0042] The present invention is a method for isolating an antibody that specifically binds to a blood group antigen-specific immunoglobulin, and the antibody can be separated through the following steps.

[0043] A first step of preparing red blood cell beads; a second step of binding IgM to the red blood cell beads; a third step of treating the red blood cell bead-IgM with an Fv-antibody library; a fourth step of separating the red blood cell bead-IgM treated in the third step; and a step of purifying the Fv bound to the red blood cell bead-IgM separated in the fourth step.

[0044] Additionally, the red blood cell beads of the first step can be manufactured through the following steps.

[0045] Step 1-1 of extracting red blood cells from an object; Step 1-2 of treating the red blood cells with glutaraldehyde; Step 1-3 of freeze-drying the red blood cells treated in Step 1-2; and Step 1-4 of crushing the freeze-dried red blood cells in Step 1-3 into a predetermined size.

[0046] Here, the subject may include various animals having blood, such as humans, mice, monkeys, etc.

[0047] Hereinafter, antibodies, antigen-binding fragments thereof or peptides that specifically bind to blood group antigen-specific immunoglobulins according to the present invention will be described through examples.

[0048] Example 1. Screening of mimotopes against type A and type B antigens from an Fv library.

[0049] It is well known that IgM, which has binding affinity for type A and type B antigens on RBCs, induces RBC agglutination when plasmas with incompatible ABO blood types are mixed. In the present invention, Fv antibodies having binding affinity for IgM specific for type A and type B antigens were selected from an Fv library. Since the selected Fv exhibits binding to IgM specific for type A antigens (and type B antigens), it is expected that the selected Fv has a function corresponding to the mimotope of type A and type B antigens on RBCs, and that the selected Fv (mimotope) can effectively prevent RBC agglutination.

[0050] The Fv library was prepared as described previously. The Fv antibody was composed of the VH of immunoglobulin G (IgG) consisting of three CDR3 regions and framework regions (FRs) (see Table 1). For the preparation of the Fv library, the amino acid sequence of the CDR3 region (11 residues) was randomized by site-directed mutagenesis. The diversity of the Fv library was estimated to be more than 105 clones / library. The Fv library was expressed on the outer membrane of E. coli using surface expression (autodisplay) technology. The high expression yield was 10 5 It was estimated that Fv of E. coli was higher than that of E. coli, and it was estimated that more than 90% of E. coli expressed Fv antibodies on their surface along with the total number of E. coli.

[0051] RegionOligonucleotide sequence (5' → 3')Amino acid sequence (N-term → C-term)Frame-1 (FR1)GAA GTG CAG CTC GTG GAA AGC GCT GCC GAA GTT CGG CGT CCT GGG GCT AGC GTG AAG ATC ACC TGC AAA GCG TCC GGC TAT TCA TTC AGC (SEQ ID NO: 11)EVQLVESAAEVRRPGASVKITCKASGYSFS (SEQ ID NO: 1)CDR1ACC TAT GGG ATT CAG (SEQ ID NO: 12)TYGIQ (SEQ ID NO: 2)Frame-2 (FR2)TGG ATG CGC CAA GCG CCA GGC CAG CGT CCG GAA TGG CTT GGG (SEQ ID NO: 13)WMRQAPGQRPEWLG (SEQ ID NO: 3)CDR2TGG ATA CAT GCA GGC ACA GGT GGG ACT AAG TAC TCG CGC AAA TTT CAG GGT (SEQ ID NO: 14)WIHAGTGGTKYSRKFQG (SEQ ID NO: 4)Frame-3 (FR3)CGC ATT ACT ATC ACC CGT GAT ACC AGC GCG AAT ACC GTC TAT CTG GAT CTG AAC TCT CTG ACA TCG GAG GAT ACG GCC GTC TAT TAT TGC GCT CGT (SEQ ID NO: 15)RITITRDTSANTVYLDLNSLTSEDTAVYYCAR (SEQ ID NO: 5)CDR3 (template)GAC AAA GTT ACA GTC TGG GCT TGT CAG GAT AAT (SEQ ID NO: 16)DKVTVWACQDN (SEQ ID NO: 6)Frame-4 (FR4)TGG GGT CAA GGT ACT ACG GTT ACG GTC AGC AGT (SEQ ID NO: 17)WGQGTTVTVSS (SEQ ID NO: 7)

[0052] For screening of the Fv library, purified IgM specific for type A and type B antigens was prepared from type AB human plasma using RBC beads. As illustrated in Figure 1, RBC beads were prepared by the steps of (1) glutaraldehyde treatment, (2) lyophilization, and (3) grinding into microbeads with a diameter of approximately 50-100 μm. Isolation of IgM specific for type A (and type B) antigens was performed from type AB human plasma and crude IgM extracts from Sihdia Co (Singyang Diagnostics, Korea) using RBC beads.

[0053] Mithopone screening of RBCs for type A and B antigens was performed using an Fv library containing purified RBCs bound to IgM. Because IgM is an octamer of IgGs, it may have an empty binding pocket even after binding to RBCs. In the first step, IgM-bound RBCs were prepared by mixing an excess of purified IgM specific for type A (and B) antigens with RBCs harboring type A antigens. As shown in Figure 2(a), the IgM-bound RBCs were mixed with the Fv library. Unbound E. coli cells and RBCs from the Fv library were separated by centrifugation. The separated RBCs were cultured on an agar plate, and the bound E. coli were able to form colonies on the agar plate, indicating that they had binding affinity for the IgM bound to the RBCs. Several clones were selected from the agar plate and the binding affinity of a specific type of antigen to RBCs was analyzed using dynamic laser scattering (DLS). Typically, DLS measures the scattering of monochromatic light to determine the hydrodynamic radius of a particle. To measure the affinity of E. coli to RBCs, cultured E. coli from different colonies (clones) were reacted with type A RBCs (blue), type B RBCs (red), and PBS (black). When E. coli binds to RBCs with an average diameter of 10 μm, an increase in the size of the RBCs can occur, and this change can be observed by DLS.

[0054] For type A, as shown in Fig. 2(b), five clones were observed to show size changes after reaction with type A RBCs (blue). No significant changes in DLS signals occurred with type B RBCs (red) and PBS (black). After genetic analysis of the CDR3 region of the Fv antibody, only one clone (clone number 28) was determined to have a valid mutant sequence, while the other four clones were determined to have genetic sequences identical to the template sequence.

[0055] For type B, as shown in Fig. 2(c), four clones were observed to show size changes after reaction with type B RBCs (red). No significant changes in DLS signals occurred with type A RBCs (blue) and PBS (black). After genetic analysis of the CDR3 region of the Fv antibody, two clones (clone numbers 6 and 22) were determined to have valid mutant sequences, and the other two clones were determined to have genetic sequences identical to the template sequence. The oligonucleotide sequences and amino acid sequences of the CDR3 region for the selected clones are shown in Table 2.

[0056] CloneCDR3 Oligonucleotide sequence (5' → 3')CDR3 Amino acid sequence (N-term → C-term)TemplateGAC AAA GTT ACA GTC TGG GCT TGT CAG GAT AAT (SEQ ID NO: 16)DKVTV WACQD N (SEQ ID NO: 6)A28TGT CGT GTT GTC CTG GAG GAT ATT ATG GAT TAC (SEQ ID NO: 18)CRVVL EDIMD Y (SEQ ID NO: 8)B6GAT GGG CCT GTC AGG AGG ACT ACA GGT GAT GTC (SEQ ID NO: 19)DGPVR RTTGD V (SEQ ID NO: 9)B22GGT CGG GGT AGT GCC CGG CAA GGT GTA GAT TAT (SEQ ID NO: 20)GRGSA RQGVD Y (SEQ ID NO: 10)

[0057] The selected Fv antibodies were expressed as fusion proteins with GFP. As shown in Fig. 3(a), the Fv antibodies were composed of three CDRs including the screened CDR3 region and four FRs. The expression of the three Fv antibodies was observed to have a molecular weight of 40.3 kDa from SDS-PAGE. The binding constants (KD) of the three expressed Fv antibodies against IgM were estimated using an SPR biosensor. As shown in Fig. 3(b), IgM specific for type A antigens (and type B antigens) was immobilized on the gold surface of the SPR biosensor, and the expressed Fv antibodies were injected in the concentration range of 0.62 to 50 nM, and then the association-dissociation profiles were recorded according to the concentration of the expressed Fv antibodies, as shown in Fig. 3(c). The association constant (K D ) was calculated to be 12 nM (R2=0.960, n=3) for A28 Fv, 10 nM (R2=0.955, n=3) for B6 Fv, and 6.3 nM (R2=0.938, n=3) for B22 Fv. These results indicate that (1) Fv-antibodies can strongly bind to IgM against not only B but also A antigens with high affinity in the nanomolar range, and (2) Fv-antibodies can be used as mimotopes for both A and B antigens. Type A IgM: K D = 130nM, type B IgM: K D = 60nM

[0058] Example 2. Evaluation of RBC aggregation inhibition activity of Fv antibodies

[0059] The screening and expression of Fv antibodies were aimed at identifying mimotopes of type A and type B antigens on RBCs, and possessed binding affinity to IgM specific for these antigens. Mimotope activity was assessed by estimating the inhibition of RBC agglutination induced by mixing plasma with IgM of ABO-mismatched types. RBC agglutination was semiquantitatively estimated using a gel card from BioRad Diagnostics (Hercules, CA, USA). The gel card consisted of a gel filtration resin impenetrable to aggregated RBCs.

[0060] The gel card experiment was conducted through the following process.

[0061] A. 1 / 2 7 50 μL of diluted type A anti-sera (Sihdia Shingyang Diagnostics, Siheung, Korea) and 50 μL of A28 Fv (0-24.8 μM) were mixed in a 1 / 2 ratio. 7 50 μL of diluted B-type anti-sera (Sihdia Shingyang Diagnostics, Siheung, Korea) and 50 μL of B6 Fv, B22 Fv (0-24.8 μM) were reacted on a rotator at room temperature at 15 rpm for 1 hour.

[0062] B. For purified IgM, react 50 μL of type A IgM 150 nM and 50 μL of A28 Fv (0-24.8 μM) with 50 μL of type B IgM 150 nM and 50 μL of B6 Fv, B22 Fv (0-24.8 uM) on a rotator at room temperature at 15 rpm for one hour.

[0063] C. To further confirm the selectivity of Fv, 1 / 2 7 Diluted anti-sera and purified IgM are mixed with Fv antibodies and reacted at 15 rpm on a rotator at room temperature for one hour.

[0064] D. 1 / 2 750 μL of diluted type A anti-sera and 50 μL of A28 peptide (0-250 μM), 1 / 2 7 50 μL of diluted B-type anti-sera and 50 μL of B6 peptide and B22 peptide (0-24.8 μM) were reacted on a rotator at room temperature at 15 rpm for 1 hour.

[0065] E. For purified IgM, react 50 μL of 150 nM type A IgM and 50 μL of A28 peptide (0-250 μM) with 50 μL of 150 nM type B IgM and 50 μL of B6 peptide and B22 peptide (0-24.8 μM) on a rotator at room temperature at 15 rpm for one hour.

[0066] F. Combine 1 mL of 0.8% Affirmagen A1 and B cells (Ortho-Clinical Diagnostics, Pencoed, UK) and 1 mL of ID-Diluent 2 (BioRad Diagnostics, Hercules, CA, USA) and mix gently using a pipette.

[0067] G. Add 25 μL of the prepared samples and 50 μL of the prepared RBCs to a gel card (ID-Card NaCl, Enzyme Test and Cold Agglutinins) (BioRad Diagnostics, Hercules, CA, USA) and centrifuge at 1030 rpm for 10 minutes using ID-Centrifuge 12 S II (BioRad Diagnostics, Hercules, CA, USA).

[0068]

[0069] Aggregated RBCs were located at the top of the gel filtration resin (red). For non-aggregated RBCs, perforated RBCs (red) were observed at the bottom of the gel card tube. Depending on the aggregation rate, perforated RBCs (red color) were observed in the middle of the tube, and the aggregation rate could be scored semiquantitatively.

[0070] The inhibitory activity of the three expressed Fv antibodies was estimated by mixing different concentrations of expressed Fv antibodies with a mixture of RBCs and mismatched anti-sera. As shown in Figure 4(a), a mixture of anti-sera (specific for type A antigens and containing IgM specific for type A antigens) and RBCs (containing type A antigens) was prepared, and the mixture was treated with different concentrations of Fv antibody (A28 Fv). At low concentrations of Fv antibody (lane 1), RBC agglutination was observed through red RBCs on the top of the gel filtration resin. As the concentration of Fv antibody increased, non-aggregated RBCs were found between the top and bottom of the tube. At sufficiently high concentrations of Fv antibody (lane 6), RBC spots (red) were observed at the bottom of the tube. These results demonstrated that the Fv antibody (A28 Fv) could inhibit the agglutination of RBCs by a mismatched anti-sera (specific for class A antigens and containing class A antigen-specific IgM). Therefore, the expressed Fv antibody (A28Fv) could be used as a mimetic for class A antigens and could be used to inhibit the activity of class A antigen-specific IgM.

[0071]

[0072] In addition, inhibition assays for mimotopes of B antigens were performed by treating mixtures of anti-sera (specific for B antigens and containing B antigen-specific IgM) and RBCs (containing B antigens) with different concentrations of Fv antibodies (B6Fv and B22Fv). As shown in Fig. 4(a), in the case of sufficiently high concentrations of Fv antibodies (lane 6), aggregated RBCs were found on top of the gel filtration resin (lane 1). These results indicate that the expressed Fv antibodies (B6 Fv and B22 Fv) can be used as mimotopes of B antigens and can be used to inhibit the activity of IgM specific for B antigens.

[0073] The selectivity of the mimotope was estimated by measuring the IgM inhibition rate of mismatched ABO antigens. For example, when the mimotope for type A antigen (A28 Fv) was treated with a mixture of antisera (containing IgM specific for type B antigens and specific for type B antigens) and RBCs (containing type B antigens), RBC agglutination was observed. As shown in Figure 4(b), RBC agglutination was observed even at high concentrations of the mimotope for type A antigen (A28 Fv), which was prevented from agglutination by the mimotope. The same selectivity was observed with the mimotopes for type B antigens (B6 Fv and B22 Fv). These results demonstrated that the mimotope for type A and B antigens can bind to IgM with high affinity, prevent RBC agglutination, and exhibit high selectivity for each IgM specific for type A and B antigens, respectively.

[0074] To confirm that the inhibitory activity arose from IgM within the antisera, an inhibition assay was performed using purified IgM rather than whole antisera. The inhibitory activity of the three synthesized Fvs was measured by mixing Fv antibodies expressed at different concentrations with a mixture of mismatched IgM instead of RBCs and antisera. As shown in Figure 5(a), a mixture of purified IgM (specific for class A antigens) and RBCs (containing class A antigens) was prepared, and the mixture was treated with different concentrations of Fv antibody (A28 Fv). At low concentrations of Fv antibody (A28 Fv) (lane 1), RBC agglutination was observed through red RBCs on the top of the gel filtration resin. As the concentration of Fv antibody increased, non-aggregated RBCs were found between the top and bottom of the tube. At sufficiently high concentrations of Fv antibody (lane 6), RBC spots (red) were observed at the bottom of the tube. These results showed that the agglutination of RBCs (containing type A antigens) by mismatched IgM (specific for type A antigens) was prevented by the inhibition of IgM (specific for type A antigens) by mimotope (A28 Fv). In addition, the inhibition assay for mimotope of type B antigens was performed by treating the mixture of IgM (specific for type B antigens) and RBCs (containing type B antigens) with different concentrations of Fv antibodies (B6Fv and B22Fv). As shown in Fig. 5(a), in the case of sufficiently high concentrations of Fv antibodies (lane 6), aggregated RBCs were found on the upper side of the gel filtration resin (lane 1). These results showed that the expressed Fv antibodies (B6Fv and B22Fv) could inhibit the activity of IgM (specific for type B antigens).

[0075] The selectivity of the mimotope was estimated by measuring the IgM inhibition rate against mismatched ABO antigens. For example, a mimotope for type A antigen (A28 Fv) was treated with a mixture of IgM (against type B antigen) and RBCs (using type B antigen), and RBC agglutination was observed. As shown in Figure 5(b), RBC agglutination was observed even with a high concentration of mimotope for type A antigen (A28 Fv). The same selectivity was observed with mimotopes for type B antigens (B6 Fv and B22 Fv). These results demonstrate that mimotopes for type A and B antigens can bind to IgM with high affinity, prevent RBC agglutination, and exhibit high selectivity for each IgM specific for type A and B antigens, respectively.

[0076] Example 3. Evaluation of RBC aggregation inhibition activity of peptides

[0077] The selected Fv antibodies were confirmed to inhibit the activity of IgM specific for type A and type B antigens for RBC agglutination. As mentioned above, the Fv library was generated by site-directed mutagenesis of the CDR3 region, and the binding of the selected Fv antibodies occurred through the CDR3 region. To determine whether the binding of the CDR3 region could inhibit RBC agglutination, the CDR3 region in Table 2 was synthesized as a peptide (15 residues) and the inhibitory activity was estimated. The inhibitory activity of the three expressed Fv antibodies was estimated by mixing different concentrations of the synthetic CDR3 peptides with a mixture of RBCs and mismatched anti-sera. As shown in Figure 6(a), a mixture of anti-sera (specific for type A antigens and containing type A antigen-specific IgM) and RBCs (containing type A antigens) was prepared, and the synthesized CDR3 peptide (A28 peptide) was treated with different concentrations. At low concentrations of the synthesized CDR3 peptide (A28 peptide) (lane 1), RBC agglutination was observed via red RBCs at the top of the gel filtration resin. When the concentration of the synthesized CDR3 peptide (A28 peptide) was increased, non-aggregated RBCs were found between the top and bottom of the tube. When the concentration of the synthesized CDR3 peptide (lane 6) was sufficiently high, RBC spots (red) were observed at the bottom of the tube. These results demonstrated that the synthesized CDR3 peptide (A28 peptide) could be used to inhibit RBC agglutination by mismatched antisera (specific for class A antigens and including class A antigen-specific IgM). Therefore, the synthesized CDR3 peptide (A28 peptide) can be used as a mimotope for class A antigens and to inhibit IgM (specific for class A antigen) activity.In addition, inhibition assays for mimotopes of B antigens were performed by treating a mixture of antisera (specific for B antigens and containing IgM specific for B antigens) and RBCs (containing B antigens) with different concentrations of the synthesized CDR3 peptides (B6 peptide and B22 peptide). As shown in Fig. 6(a), when the concentration of the synthesized CDR3 peptides (lane 6) was sufficiently high, aggregated RBCs were found on top of the gel filtration resin (lane 1). These results indicate that the expressed CDR3 peptides (B6 peptide and B22 peptide) can be used as mimotopes of B antigens and can be used to inhibit IgM (specific for B antigen) activity.

[0078] To confirm that the inhibitory activity arose from purified IgM in the antisera, an inhibition assay was performed using purified IgM rather than whole antisera. The inhibitory activities of the three synthesized IgMs were estimated by mixing different concentrations of the synthesized CDR3 peptides with a mixture of RBCs and mismatched IgM instead of antisera. As shown in Figure 6(b), a mixture of purified IgM (against type A antigen) and RBCs (with type A antigen) was prepared and treated with different concentrations of the synthesized CDR3 peptide (A28 peptide). At low concentrations of the synthesized CDR3 peptide (A28 peptide) (lane 1), RBC agglutination was observed, as evidenced by red RBCs at the top of the gel filtration resin. As the concentration of Fv antibody increased, non-aggregated RBCs were found between the top and bottom of the tube. When the concentration of the synthesized CDR3 peptide (lane 6) was sufficiently high, RBC spots (red) were observed at the bottom of the tube. These results showed that the agglutination of RBCs (containing type A antigens) by mismatched IgM (specific for type A antigens) was prevented by the inhibition of IgM (specific for type A antigens) by the mimotope (A28 peptide). In addition, an inhibition assay for the mimotope of type B antigens was performed by treating a mixture of IgM (specific for type B antigens) and RBCs (containing type B antigens) with different concentrations of the synthesized CDR3 peptides (B6 peptide and B22 peptide). As shown in Figure 6(b), in the case of a sufficiently high concentration of Fv antibody (lane 6), aggregated RBCs were found on top of the gel filtration resin (lane 1). These results showed that the synthesized CDR3 peptides (B6 peptide and B22 peptide) could inhibit IgM (specific for type B antigen) activity.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to a blood group antigen-specific immunoglobulin, comprising a CDR3 selected from the group consisting of a CDR3 comprising an amino acid sequence of SEQ ID NO: 8, a CDR3 comprising an amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising an amino acid sequence of SEQ ID NO:

10.

2. In claim 1, An antibody or antigen-binding fragment thereof, wherein the blood group antigen-specific immunoglobulin is a type A antigen-specific IgM or a type B antigen-specific IgM.

3. A peptide that specifically binds to a blood group antigen-specific immunoglobulin, comprising a peptide selected from the group consisting of a peptide comprising the amino acid sequence of SEQ ID NO: 8, a peptide comprising the amino acid sequence of SEQ ID NO: 9, and a peptide comprising the amino acid sequence of SEQ ID NO:

10.

4. In claim 3, A peptide wherein the blood group antigen-specific immunoglobulin is a type A antigen-specific IgM or a type B antigen-specific IgM.

5. A nucleic acid encoding an antibody or antigen-binding fragment thereof according to claim 1 or claim 2, or a peptide according to claim 3 or claim 4.

6. A recombinant expression vector comprising a nucleic acid according to claim 5.

7. A cell transformed with a recombinant expression vector according to claim 6.

8. In claim 7, The above cell is a cell selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli, and insect cells.

9. A method for producing an antibody, an antigen-binding fragment thereof, or a peptide comprising the following steps: (a) a step of culturing the cell of claim 7; and (b) a step of recovering an antibody, an antigen-binding fragment thereof, or a peptide from the cultured cells and / or their culture medium.

10. A pharmaceutical composition for preventing or treating an immune disease, comprising an antibody or an antigen-binding fragment thereof according to claim 1 or claim 2, or a peptide according to claim 3 or claim 4 as an active ingredient.

11. In claim 10, A composition, wherein the above immune disease comprises a disease selected from the group consisting of transplant rejection disease, transplant disease, and fibrosis.

12. In claim 10, A composition comprising an antibody, an antigen-binding fragment thereof, or a peptide comprising the amino acid sequence of the above sequence number 8, which suppresses an immune response of blood or an organ containing a type A antigen.

13. In claim 10, A composition comprising an antibody, an antigen-binding fragment thereof, or a peptide comprising the amino acid sequence of the above sequence number 9 or sequence number 10, which suppresses an immune response of blood or an organ containing a type B antigen.

14. Step 1-1: Extracting red blood cells from an object; Step 1-2 of treating the above red blood cells with glutaraldehyde; Step 1-3 of freeze-drying the processed red blood cells of Step 1-2; and A method for manufacturing red blood cell beads, comprising a step 1-4 of crushing the freeze-dried red blood cells of the above steps 1-3 into a predetermined size.

15. A first step of preparing a red blood cell bead according to claim 14; A second step of binding IgM to the above red blood cell beads; A third step of processing the above red blood cell bead-IgM into an Fv-antibody library; A fourth step of separating the red blood cell bead-IgM processed in the third step; and A method for isolating an antibody that specifically binds to a blood group antigen-specific immunoglobulin, comprising a step of purifying Fv bound to the separated red blood cell bead-IgM of the fourth step.

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