TW202041862A - Antibody and antibody fragments, kit and method for detecting miltenberger blood group antigen - Google Patents

Abstract

The disclosure provides an isolated antibody or antibody fragment that specifically binds an epitope of Miltenberger blood group antigen. Wherein the epitope is a part of an amino acid sequence which is selected from the group consisting of SEQ ID NO.1 to SEQ ID NO.6, and which at least includes the amino acid sequence of “Arg-Asp-Thr-Tyr”. The disclosure further provides a kit and a method for detecting Miltenberger blood group antigen.

Description

Antibody, fragment, kit and method for detecting Yonetianbao blood group antigen

The invention relates to an antibody and fragments thereof, a kit and a method for detecting Yonetianbao antigen.

Second only to the Rh blood group system, there is a special Miltenberger sub-blood group system in the MNS blood group system. The blood group antigen is a red blood cell phenotype of a series of low-frequency antigen combinations identified by human serum immune antibodies. Miltenberger blood group antigen (Miltenberger blood group antigen) is very likely to be derived from homologous blood group glycoprotein A (GYPA), blood group glycoprotein B (glycophorin B, GYPB), and blood group in the process of evolution. A hybrid protein produced by genetic recombination of glycophorin E (GYPE). Currently, 11 subtypes of Mitianbao blood group antigen have been found, including Mitianbao blood group antigen type I (Mi.I) to Mitianbao blood group antigen type eleven (Mi.XI). Mi Tianbao blood group antigen type III (Mi.III) is speculated to be a section of glycoprotein A gene inserted into glycophorin B through homologous recombination. Therefore, the gene sequence of the third type of Yonetianbao blood group antigen is in the form of a blood group glycoprotein BAB mixed gene. This BAB mixed gene transcribes a strong antigenic Mur peptide, so the third type of Yonetianbao blood group antigen is also called Gp. Mur (see, for example, Journal of Molecular Evolution (1995) 41:4, 478-486).

In Taiwan, Mitianbao blood group antigen type 3 has the highest frequency of all special blood types; especially in certain indigenous ethnic groups, its frequency is as high as 20 to 90%. Overall, there are about 6.27% of Taiwanese people who are positive for the third type of blood group antigen. Mitianbao blood group antigen type III occurs in certain Southeast Asian countries with a frequency equivalent to Taiwan, such as Thailand in 9-10%, Philippines in 8.3%, Hong Kong in 6.3%, Indonesia in 2.47% and China in 1.9%. However, the frequency of the third type of the Yonetianbao blood group among Caucasians (Europe, America, Australia, etc.), Northern Han Chinese and Japanese is less than 0.01%.

The clinical report confirmed that Yonetianbao blood group antigen type III can cause allogeneic immune diseases, such as adverse blood transfusion reactions and neonatal hemolysis. Therefore, it is necessary to determine whether the donor's blood contains Yadapu antigen before blood transfusion to avoid the aforementioned adverse reactions. However, there are currently no commercially available monoclonal antibodies and reagents against the Yonetianbao blood group antigen, and there is no suitable and rapid detection method.

Therefore, there is still an urgent need for an antibody, kit and method for detecting whether the donor's sample has the Yonetianbao blood group antigen, which can confirm whether the donor and the recipient's Yonetianbao blood type match before blood transfusion to avoid adverse reactions happened.

In view of the above-mentioned problems, the purpose of the present invention is to provide an antibody and fragments thereof, kits and methods for detecting Yonetianbao blood group antigen. It is used before blood transfusion to confirm whether the donor's sample contains Yadapu blood group antigen, so as to screen the donor sample suitable for the recipient, and avoid the adverse reaction of alloimmune diseases after the sample is transferred to the recipient.

To achieve the above objective, the present invention provides an isolated antibody or fragment thereof, the antibody or fragment thereof selectively binds specifically to an epitope of the Miltenberger blood group antigen, wherein the epitope is selected from SEQ ID NO. Part of the amino acid sequence in the group consisting of 1 to SEQ ID NO. 6, and at least includes the amino acid sequence Arg-Asp-Thr-Tyr.

In one embodiment, the antibody or fragment thereof includes: a heavy chain variable region, including CDR-H1 having the amino acid sequence of SEQ ID NO. 7, CDR-H2 having the amino acid sequence of SEQ ID NO. 8, and CDR-H3 of the amino acid sequence of SEQ ID NO.9; and a light chain variable region, including CDR-L1 having the amino acid sequence of SEQ ID NO.10, and CDR having the amino acid sequence of SEQ ID NO.11 -L2 and CDR-L3 with the amino acid sequence of SEQ ID NO.12.

To achieve the above objective, the present invention further provides an isolated antibody or fragment thereof, which has a binding domain and includes: a heavy chain variable region, including CDR-H1 with the amino acid sequence of SEQ ID NO. 7 and SEQ ID NO. .8 CDR-H2 with amino acid sequence and CDR-H3 with amino acid sequence of SEQ ID NO.9; and a light chain variable region including CDR-L1 with amino acid sequence of SEQ ID NO.10 CDR-L2 with the amino acid sequence of SEQ ID NO.11 and CDR-L3 with the amino acid sequence of SEQ ID NO.12.

In one embodiment, the heavy chain variable region has the amino acid sequence of SEQ ID NO.13.

In one embodiment, the light chain variable region has the amino acid sequence of SEQ ID NO.14.

In one embodiment, the heavy chain variable region is encoded by the nucleotide sequence of SEQ ID NO.15.

In one embodiment, the light chain variable region is encoded by the nucleotide sequence of SEQ ID NO.16.

In one embodiment, the antibodies or fragments thereof are selected from the group consisting of monoclonal antibodies, humanized antibodies, recombinant antibodies, human antibodies, biologically active fragments of antibodies, antibody mimics, and any combination thereof.

In one embodiment, the antibody or fragment thereof is selected from the group consisting of Fab, Fab', F(ab') ₂ , Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies, and any combination thereof.

To achieve the above objective, the present invention further provides a kit for detecting a Miltenberger blood group antigen in a sample of a living object, the kit including an antibody or fragment thereof as described above.

In one embodiment, the sample is whole blood or isolated red blood cells.

In one embodiment, the biological system is human.

To achieve the above objective, the present invention further provides a method for detecting a Miltenberger blood group antigen in a living object. The method includes: preparing a sample of the organism; and adding an antibody or fragment thereof as described above to In the sample, to detect whether the sample has Miltenberger blood group antigen.

In one embodiment, the sample is whole blood or isolated red blood cells.

In one embodiment, the biological system is human.

Based on the above, the effect of the present invention is that by providing an antibody and fragments thereof, kits and methods for detecting Yonetianbao blood group antigens, it is possible to confirm whether there are Yonetianbao blood group antigens in the samples of organisms for screening suitable For example, but not limited to blood transfusion samples, to avoid the adverse reactions of allogeneic immune diseases after the sample is transferred to another organism.

Hereinafter, preferred embodiments of antibodies and fragments thereof, kits and methods for detecting Yonetianbao blood group antigens according to the present invention will be described with reference to related drawings, wherein the same elements will be described with the same reference symbols.

The antibodies, fragments, kits, and methods of the present invention can be used to detect whether a sample of an organism has a Yadapu blood group antigen, and then screen a sample suitable for another organism to avoid the occurrence of allogeneic immunoreactive diseases. The following examples are used to illustrate the antibodies and fragments thereof, kits and methods for detecting Yonetianbao blood group antigens of the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as understood by those skilled in the art to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the test experiments of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used. It should be understood that the purpose of the terms used herein is only for describing specific embodiments, rather than limiting the present invention.

The term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to a specific epitope on an antigen. The antibody may be a whole immunoglobulin derived from a natural source or from a recombinant source, and may be an immunoreactive part of the whole immunoglobulin. Antibodies useful in the present invention can exist in various forms, including, for example, monoclonal antibodies, humanized antibodies, recombinant antibodies, human antibodies, biologically active fragments of antibodies, and antibody mimics. Antibodies can be from natural sources or from recombinant sources. Antibodies are usually tetramers of immunoglobulin molecules. The "antibody" provided herein includes any immunoglobulin type (such as IgG, IgM, IgD, IgE, IgA and IgY), any type (such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subtype (such as IgG2a) And IgG2b) members.

The term "antibody fragment" refers to at least a part of a complete antibody or a recombinant variant thereof, and refers to an antigen-binding domain, such as the epitope variable region of a complete antibody, which is sufficient to give the antibody fragment discrimination and target specificity (such as Antigen) binding capacity. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') ₂ and Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies such as sdAb (VL or VH), VHH domains, and antibody fragments The formation of multi-specific antibodies. Fab fragments (antigen binding) are antibody fragments produced by papain digestion of full-length immunoglobulins, or fragments with the same structure produced synthetically (for example, by recombination methods). The Fab fragment contains the variable region of the light chain (containing VL and CL) and a constant region (CH1) containing the variable region of the heavy chain (VH) and the heavy chain. F(ab') ₂ fragments are antibody fragments produced by pepsin digestion of immunoglobulins at pH 4.0 to 4.5, or fragments with the same structure produced synthetically (for example, by recombination methods). The F(ab') ₂ fragment basically contains two Fab fragments, where each heavy chain part contains a few additional amino acids, including cysteine residues that form a disulfide bond connecting the two fragments. Fab' fragments are fragments containing half (one heavy chain and one light chain) F(ab') ₂ fragments. Fv fragments are fragments that only contain the light chain variable region (VL) and heavy chain variable region (VH) of an antibody molecule. The scFv fragment refers to an antibody fragment that contains a light chain variable region (VL) and a heavy chain variable region (VH) covalently linked by a polypeptide linker in any order. The linker connects the two variable regions without disturbing their length. Exemplary linkers are (Gly-Ser)n residues, some Glu or Lys residues are dispersed throughout the residue to increase solubility.

The term "heavy chain variable region (VH)" refers to a functional region and a non-functional region comprising at least a part of the complementarity determining region (CDR), wherein the functional region of the heavy chain variable region alone or with another antibody domain (such as light The chain variable region (VL)) combination can bind to the antigen. An exemplary functional region of the heavy chain variable region in the present invention is a heavy chain variable region containing CDR-H1, CDR-H2 and CDR-H3.

The term "light chain variable region (VL)" refers to a functional region and a non-functional region (FR) comprising at least a part of the complementarity determining region (CDR), wherein the functional region of the light chain variable region is alone or in combination with another antibody domain (For example, heavy chain variable region (VH)) combination can bind to antigen. The exemplary functional region of the light chain variable region in the present invention is the light chain variable region containing CDR-L1, CDR-L2 and CDR-L3.

The term "monoclonal antibody" refers to the same monoclonal antibody population, which means that each individual antibody molecule in the monoclonal antibody population is the same as other antibody molecules. Monoclonal antibodies can be produced by many known methods (for example, J. Clin. Pathol. (2004) 57, 912-917; and J Clin Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be produced by immortal B cells, such as by fusion with myeloma cells to produce fusion tumor cell lines or by infecting B cells with viruses such as EBV, or by using recombinant technology in vitro by A plastid with artificial nucleotide sequences encoding antibodies is used to transform host cells to produce antibodies from the host cells.

Humanized antibodies can be produced from any suitable mammalian source capable of producing antibodies using conventional methods, including but not limited to mice, rats, camels, llamas, rabbits or other vertebrates.

The term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as an antibody expressed by a phage or yeast expression system. The term should also be interpreted as an antibody produced by a DNA molecule encoding an antibody through a synthetic method, and the DNA molecule will express the antibody protein or the amino acid sequence of the designated antibody. Synthetic DNA or DNA molecules known in the art can be used. Amino acid sequence technology to obtain DNA or amino acid sequence.

The DNA sequences of human antibodies, especially the complementarity determining regions (CDR), can be isolated according to procedures well known in the art. Preferably, as described in International Patent Application Publication No. WO 1987/02671, the DNA sequence of human CDRs is isolated from immortal B cells. The CDRs suitable for producing the antibody of the present invention can be obtained in a similar manner from DNA encoding a monoclonal antibody capable of binding to the target molecule. The terms "complementarity-determining region" and "CDR" are used interchangeably, and refer to one of a plurality of parts in each variable region that together form the antigen-binding domain of an antibody. Each variable region contains three CDRs, called CDR1, CDR2, and CDR3. The three CDRs are not connected along the linear amino acid sequence, but are close in the folded polypeptide. The CDR is located in a loop connecting the parallel strands of the beta sheet of the variable region. As described herein, those skilled in the art can identify CDRs based on Kabat or Chothia numbering (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917).

The term "Yitianbao blood group antigen" refers to the sub-blood group system in the MNS blood group system. Nowadays, 11 subtypes (subtypes) have been discovered, including "Yonetianbao blood group antigen" Type I (Mi.I) to Mitianbao blood group antigen No. 1 Eleven type (Mi.XI). "Yetianbao blood group antigen" is composed of at least one of Mia, Vw, Mur, Hut, Hil, Hop, Nob, DANE, TSEN, MINY, and MUT antigens. Among them, "Yitianbao blood group antigen type III" includes Mia, Mur, Hil, MINY and MUT antigens (see, for example, ISBT Science Series (2011) 6, 296-301).

The term "antigen" refers to a molecule that causes an immune response. This immune response may involve the production of antibodies, the activation of specific immunocompetent cells, or both. Those skilled in the art will understand that any macromolecule, including almost all proteins or peptides, can be used as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that triggers an immune response encodes the term "antigen" herein. In addition, those skilled in the art understand that the antigen does not need to be encoded by the nucleotide sequence of the entire gene. It is obvious that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art understand that an antigen does not need to be encoded by a "gene". It is obvious that the antigen can be synthetic or can be obtained from a biological sample. The aforementioned biological samples may include, but are not limited to, tissue samples, tumor samples, cells or biological fluids.

The term "epitope" refers to a small chemical molecule on an antigen that can trigger an immune response and induce a B and/or T cell response. The antigen can have one or more epitopes. Most antigens have multiple epitopes; that is, these antigens are multivalent. Generally, the epitope size is about 10 amino acids and/or sugars. Preferably, the epitope is about 4 to 18 amino acids, more preferably about 5 to 16 amino acids, even more preferably 6 to 14 amino acids, more preferably about 7 to 12 amino acids Acid, most preferably about 8 to 10 amino acids. Those skilled in the art understand that the main criterion for antigen specificity is usually the entire three-dimensional structure rather than the specific linear sequence of the molecule.

The term "specific binding" as used herein with respect to antibodies refers to antibodies that recognize a specific antigen but do not substantially recognize or bind to other molecules in the sample. For example, an antibody that specifically binds to an antigen from one species can also bind to an antigen from one or more species.

The term "binding domain" refers to a polypeptide domain that binds or interacts with a specific target structure/antigen/epitope in a specific manner. "Combine or produce interaction" can also be understood as the definition of a "specific identification effect". As in the present invention, the term "specific recognition" means that the antibody molecule can interact with the antigen in a specific manner with at least one, preferably at least two, and more preferably at least three epitopes of the Yadapu blood group antigen as defined herein. Interaction and/or combination.

The term "immune response" is defined as a cellular response to an antigen that occurs when lymphocytes identify antigen molecules as foreign and induce antibody formation and/or activate lymphocytes to remove the antigen.

The terms "organism", "donor" or "recipient" can be human or non-human mammals. Non-human mammals include, for example, domestic animals and pets, such as sheep, cattle, pigs, canines, cats, and murine mammals. Preferably, the organism is human.

The term "sample" refers to a sample obtained from an organism or a composition of an organism (eg, cells). The sample can be any biological tissue or liquid. Samples include, but are not limited to, bone marrow, sputum, whole blood, serum, plasma, lymph, blood cells (for example, red blood cells), urine, peritoneal fluid, and pleural fluid or cells derived from these samples. Biological samples may also include parts of tissues, such as frozen sections taken for histological purposes. Preferably, the sample is whole blood or isolated red blood cells.

The term "isolated" as used herein for nucleic acids (such as DNA or RNA) refers to a DNA or RNA molecule corresponding to other DNA or RNA that exists in the natural source of the macromolecule. The term "isolated" as used herein also refers to nucleic acids or peptides that are substantially free of cellular material, viral material or culture medium, or chemical precursors or other chemical materials when produced by recombinant DNA technology. In addition, "isolated nucleic acid" means to include non-naturally occurring nucleic acid fragments and will not be found in the natural state. The term "isolated" is also used herein to mean a polypeptide isolated from other cellular proteins, and is meant to include purified and recombinant polypeptides. "Isolated antibody" or "isolated antibody fragment" is an antibody or antibody fragment isolated from a substance secreted by a host cell, and does not contain cellular material, viral material or culture medium.

The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. The protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that the protein or peptide sequence can contain. Polypeptides include any peptide or protein that contains two or more amino acids connected to each other through peptide bonds. As used herein, the term refers to short chains, which are also commonly referred to in the art as peptides, oligopeptides, and oligomers; for example, longer chains, which are commonly referred to as proteins in the art, and there are many types . "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified Peptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides or combinations thereof.

The term "encoding" refers to the inherent characteristics of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA, having a definite nucleotide sequence (ie, rRNA, tRNA, and mRNA) or a definite amino acid sequence, Used as a template for the synthesis of other polymers and macromolecules in biological processes and the biological properties produced by this process. Therefore, in a cell or other biological system, if the transcription and translation of mRNA corresponding to the gene produces a protein, the gene encodes a protein. The coding strand, whose nucleotide sequence is the same as the mRNA sequence and is usually provided in the sequence listing, and the non-coding strand, used as a template for gene or cDNA transcription, can be referred to as an encoding protein or other encoding genes or cDNA product.

The term "allogeneic" refers to any substance derived from different animals of the same species.

The term "vector" refers to a composition of matter that contains isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides related to ionic or amphiphilic compounds, plastids, and viruses. Therefore, the term "vector" includes autonomously replicating plastids or viruses. The term should also be interpreted to include aplastic and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and the like.

"Host cells" are cells used to receive, maintain, regenerate, and amplify vectors. The host cell can also be used to express the polypeptide encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. For example, a host cell is transfected, transformed, or transduced with a vector to contain the vector for expressing a polypeptide (such as an antibody or a fragment thereof) encoded by the vector. "Host cells" can be bacteria, yeasts, bacteriophages, mammalian cells (such as but not limited to CHO, COS, 239F, SF9 and/or other cell strains) that can be used to produce the polypeptide encoded by the vector.

The term "transfected" or "transformed" or "transduced" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. "Transfected" or "transformed" or "transduced" cells are cells that have been transfected, transformed or transduced with exogenous nucleic acid. Cells include primary subject cells and their progeny.

In the context of the present invention, the following abbreviations are used to denote nucleic acid bases that are commonly present. "A" means adenosine, "C" means cytosine, "G" means guanosine, "T" means thymidine, "U" means urine Uridine.

In the context of the present invention, the following abbreviations are used to denote amino acids that are commonly present. "Ala (A)" refers to alanine, "Phe (F)" refers to phenylalanine (Phenylalanine), "Cys (C)" refers to cysteine (Cysteine), "Sec (U)" It refers to Selenocysteine, “Asp(D)” refers to Aspartate, “Asn(N)” refers to Asparagine, “Glu(E)” is Refers to Glutamate, "Gln(Q)" refers to Glutamine, "Gly(G)" refers to Glycine, "His(H)" refers to histamine (Histidine), "Leu(L)" means Leucine, "Ile(I)" means Isoleucine, "Lys(K)" means Lysine, "Pyl(O)" refers to Pyrrolysine, "Met(M)" refers to Methionine, "Pro(P)" refers to Proline, "Arg(R) )” refers to arginine, “Ser(S)” refers to serine, “Thr(T)” refers to threonine, “Val(V)” refers to valerian Amino acid (Valine), "Trp (W)" refers to Tryptophan, and "Tyr (Y)" refers to Tyrosine.

An exemplary embodiment of the present invention is described as follows:

Isolated antibody or fragment

An embodiment of the present invention provides an isolated antibody or fragment thereof (hereinafter referred to as "anti-Mia antibody"), which selectively interacts with an epitope of Miltenberger blood group antigen (hereinafter referred to as "Mia antigen") Specific binding, wherein the epitope is selected from SEQ ID NO. 1 (DXHKRDTY), SEQ ID NO. 2 (XHKRDTYA), SEQ ID NO. 3 (HKRDTYAA), SEQ ID NO. 4 (DKRDTYPA), SEQ ID A part of the amino acid sequence in the group consisting of NO.5 (KRDTYPAH) and SEQ ID NO.6 (RDTYPAHT), and at least includes the amino acid sequence Arg-Asp-Thr-Tyr (RDTY). In this embodiment, X in the amino acid sequence of SEQ ID NO. 1 and SEQ ID NO. 2 can be T, M, or K.

In this embodiment, the anti-Mia antibody includes a heavy chain variable region, including CDR-H1 with the amino acid sequence of SEQ ID NO. 7 (GYTFTNYAIH), and CDR with the amino acid sequence of SEQ ID NO. 8 (WINAGNGNTKYSQKFQG) -H2 and CDR-H3 with the amino acid sequence of SEQ ID NO.9 (ARDFRIPSLAVAEVGLDY); and a light chain variable region including CDR-L1 with the amino acid sequence of SEQ ID NO.10 (KSSQSVLYSSNNKNYLA) ID NO.11 (WASTRES) CDR-L2 with amino acid sequence and CDR-L3 with SEQ ID NO.12 (QQLYSTPLT) amino acid sequence.

In this embodiment, the heavy chain variable region may have the amino acid sequence of SEQ ID NO. 13 (QVQLVQSGAEVKKPGASVKISCKASGYTFTNYAIHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAIYYCARDFRIPSLAVAEVGLDYWGQGTLVTVSS). In the present embodiment, the heavy chain variable region is a nucleotide sequence of SEQ ID NO.15 (CAGGTCCAGCTTGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGATTTCCTGCAAGGCTTCTGGATACACCTTCACCAACTATGCTATACATTGGGTGCGCCAGGCCCCCGGACAAAGGCTTGAGTGGATGGGTTGGATCAACGCTGGCAATGGTAACACAAAATATTCACAGAAGTTCCAGGGCAGAGTCACCATTACCAGGGACACATCCGCGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAAGACACGGCTATATATTACTGTGCGAGAGATTTTCGGATACCTTCTCTAGCAGTGGCTGAGGTCGGTCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA) encoded.

In this embodiment, the light chain variable region may have the amino acid sequence of SEQ ID NO. 14 (DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQLYSTPLTFGGGTKVEIKR). In the present embodiment, the light chain variable region is a nucleotide sequence of SEQ ID NO.16 (GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCTCCAACAATAAGAACTACTTAGCTTGGTATCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATTGTATAGTACTCCGCTCACTTTCGGCGGAGGGACCAAAGTGGAGATCAAACGA) encoded.

In another embodiment of the present invention, there is provided an isolated antibody or fragment thereof (hereinafter referred to as "anti-Mia antibody") having a binding domain including: a heavy chain variable region, including SEQ ID NO. 7 (GYTFTNYAIH) CDR-H1 with amino acid sequence, CDR-H2 with amino acid sequence of SEQ ID NO.8 (WINAGNGNTKYSQKFQG) and CDR-H3 with amino acid sequence of SEQ ID NO.9 (ARDFRIPSLAVAEVGLDY); and a light chain Variable regions, including CDR-L1 with the amino acid sequence of SEQ ID NO.10 (KSSQSVLYSSNNKNYLA), CDR-L2 with the amino acid sequence of SEQ ID NO.11 (WASTRES), and amine with SEQ ID NO.12 (QQLYSTPLT) CDR-L3 of the base acid sequence.

In this embodiment, the heavy chain variable region may have the amino acid sequence of SEQ ID NO. 13 (QVQLVQSGAEVKKPGASVKISCKASGYTFTNYAIHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAIYYCARDFRIPSLAVAEVGLDYWGQGTLVTVSS). In the present embodiment, the heavy chain variable region is a nucleotide sequence of SEQ ID NO.15 (CAGGTCCAGCTTGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGATTTCCTGCAAGGCTTCTGGATACACCTTCACCAACTATGCTATACATTGGGTGCGCCAGGCCCCCGGACAAAGGCTTGAGTGGATGGGTTGGATCAACGCTGGCAATGGTAACACAAAATATTCACAGAAGTTCCAGGGCAGAGTCACCATTACCAGGGACACATCCGCGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAAGACACGGCTATATATTACTGTGCGAGAGATTTTCGGATACCTTCTCTAGCAGTGGCTGAGGTCGGTCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA) encoded.

In this embodiment, the light chain variable region may have the amino acid sequence of SEQ ID NO. 14 (DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQLYSTPLTFGGGTKVEIKR). In the present embodiment, the light chain variable region is a nucleotide sequence of SEQ ID NO.16 (GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCTCCAACAATAAGAACTACTTAGCTTGGTATCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATTGTATAGTACTCCGCTCACTTTCGGCGGAGGGACCAAAGTGGAGATCAAACGA) encoded.

In the foregoing embodiment, the source of the anti-Mia antibody may be human, orangutan, murine, rabbit, goat, chicken antibody, preferably, the anti-Mia antibody is a human antibody. Anti-Mia antibodies include, but are not limited to, a group consisting of monoclonal antibodies, humanized antibodies, recombinant antibodies, human antibodies, biologically active fragments of antibodies, antibody mimics, and any combination thereof. As mentioned above, anti-Mia antibodies can also be antigen fragments, including but not limited to Fab, Fab', F(ab') ₂ , Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies and any combination thereof. Group. Preferably, the anti-Mia antibody system monoclonal antibody. In the foregoing embodiment, the anti-Mia antibody can be of any immunoglobulin type (such as IgG, IgM, IgD, IgE, IgA, and IgY), any type (such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subtype (Such as IgG2a and IgG2b) members. Preferably, the anti-Mia antibody is an IgM antibody. Compared with other types, IgM antibodies are in the form of pentamers with larger molecular weight and higher affinity. Therefore, when performing a hemagglutination test, the agglutination can be easily observed, which is beneficial to the use of automated machines. Interpretation. The detailed description of the hemagglutination test is as follows, and will not be repeated here.

For detection Mia Antigen kit

In an embodiment of the present invention, a kit is provided for detecting Mia antigen in a sample of a living body, and the kit includes an antibody or fragment thereof (anti-Mia antibody) as described above. Among them, the detailed description of the anti-Mia antibody is as before, and will not be repeated here.

In this embodiment, the organism may be a human or a non-human mammal. Non-human mammals include, for example, domestic animals and pets, such as sheep, cattle, pigs, canines, cats, and murine mammals. Preferably, the organism is human.

In this embodiment, samples include, but are not limited to, bone marrow, sputum, whole blood, serum, plasma, lymph fluid, blood cells (for example, red blood cells), urine, peritoneal fluid, and pleural fluid or cells from these samples. Preferably, the sample is whole blood or isolated red blood cells.

For detection Mia Antigen method

In an embodiment of the present invention, a method for detecting Mia antigens in living objects is provided, including: preparing a sample of the organism; and adding an antibody or fragment thereof (anti-Mia antibody) as described above to the sample To detect whether the sample contains Mia antigen. Among them, the detailed description of the anti-Mia antibody is as before, and will not be repeated here.

In this embodiment, the organism may be a human or a non-human mammal. Non-human mammals include, for example, domestic animals and pets, such as sheep, cattle, pigs, canines, cats, and murine mammals. Preferably, the organism is human.

In this embodiment, samples include, but are not limited to, bone marrow, sputum, whole blood, serum, plasma, lymph fluid, blood cells (for example, red blood cells), urine, peritoneal fluid, and pleural fluid or cells from these samples. Preferably, the sample is whole blood or isolated red blood cells.

In this embodiment, the steps of preparing a biological sample include, but are not limited to, collecting the required sample and performing corresponding processing on the sample. For example, if the sample is whole blood, the step of preparing the sample of the organism is only to collect the blood of the organism; or, if the sample is isolated red blood cells, the step of preparing the sample of the organism includes collecting the blood of the organism and Red blood cells are separated from this blood. Herein, methods for separating red blood cells are well known to those skilled in the art, including but not limited to centrifugation, gradient centrifugation, or any other methods that can separate red blood cells from blood.

In this example, the step of adding an anti-Mia antibody to the sample to detect whether the sample has Mia antigen can be used to determine whether the sample has Mia antigen. The methods of judgment include but are not limited to hemagglutination test, immune ink Spot method, ELISA, immunoprecipitation method, serological test or any other methods known to those skilled in the art can be used to determine whether the antibody and antigen are bound.

Build coding resistance Mia Nucleic acid of antibody, containing coding anti Mia Antibody nucleic acid carrier and Secretable anti- Mia Antibody Host cell

In this example, the isolated nucleic acid encoding the anti-Mia antibody can be obtained by any method known in the art. For example, if the nucleotide sequence of the anti-Mia antibody is known, the nucleotide sequence encoding the anti-Mia antibody can be assembled from chemically synthesized oligonucleotides. The aforementioned synthesis is, for example, but not limited to, annealing and ligating oligonucleotides respectively encoding partial nucleotide sequences of anti-Mia antibodies, and then amplifying them by PCR. The polynucleotide of the present invention can also be produced from any other suitable nucleic acid source, such as an antibody cDNA library, or a cDNA library isolated from any tissue or cell expressing antibodies (for example, selected from fusion tumor cells expressing antibodies). The cDNA encoding the light chain and heavy chain of the anti-Mia antibody prepared from the fusion tumor can be obtained by standard PCR amplification or cDNA transfer technology. Regarding antibodies obtained using phage display technology, isolated nucleic acids encoding anti-Mia antibodies in the embodiments of the present invention can be obtained from various phage selections in the library.

In an embodiment of the present invention, the isolated nucleic acid encoding the anti-Mia antibody may include a nucleotide sequence encoding at least one of the following amino acid sequences or a combination thereof: SEQ ID NO. 7 (GYTFTNYAIH), SEQ ID NO.8 (WINAGNGNTKYSQKFQG), SEQ ID NO.9 (ARDFRIPSLAVAEVGLDY), SEQ ID NO.10 (KSSQSVLYSSNNKNYLA), SEQ ID NO.11 (WASTRES) and SEQ ID NO.12 (QQLYSTPLT). In another embodiment of the present invention, the isolated nucleic acid encoding the anti-Mia antibody may comprise the nucleotide sequence set forth in SEQ ID NO. 15, which is the nucleotide sequence encoding the variable region of the heavy chain. In another embodiment of the present invention, the isolated nucleic acid encoding the anti-Mia antibody may comprise the nucleotide sequence set forth in SEQ ID NO. 16, which is the nucleotide sequence encoding the light chain variable region. In another embodiment of the present invention, the isolated nucleic acid encoding the anti-Mia antibody may comprise the nucleotide sequence set forth in SEQ ID NO. 15 and SEQ ID NO. 16.

In another embodiment of the present invention, the aforementioned nucleotide sequences encoding the heavy chain variable region and the light chain variable region can be further recombined by standard recombinant DNA technology, for example, the variable region nucleotide sequence Transform into full-length antibody nucleotide sequence, Fab nucleotide sequence or scFv nucleotide sequence. In the aforementioned recombination, the nucleotide sequence encoding the variable region of the heavy chain and the variable region of the light chain can be operably linked to another nucleotide sequence, or a nucleotide fragment encoding another protein, for example Antibody constant region or linker. The term "operably linked" as used herein means that two nucleotide sequences are linked in a functional manner, for example, so that the amino acid sequence encoded by the two nucleotide sequence fragments remains in frame, or that the protein Performance under the control of the desired promoter.

By operably linking the nucleotide sequence encoding the variable region of the heavy chain to another nucleotide sequence encoding the constant region of the heavy chain (CH1, CH2, and CH3), the isolated nucleic acid encoding the heavy chain constant region can be transformed It is a nucleic acid with a full-length heavy chain. The nucleotide sequences and fragments of the constant regions of the heavy chains of mice and humans (or other mammals) are known in the art (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest , Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. In some embodiments, the heavy chain constant region is selected from IgM species. Regarding the Fab heavy chain nucleotide sequence, the nucleotide sequence encoding the heavy chain variable region may be operably linked to only another nucleotide sequence encoding the heavy chain CH1 constant region.

By operably linking the nucleotide sequence encoding the light chain variable region to another nucleotide sequence encoding the light chain constant region CL, the isolated nucleic acid encoding the light chain variable region can be converted into a full-length light chain Nucleic acid. The nucleotide sequence and fragments of the nucleotide sequence of the constant region of the mouse and human (or other mammals) light chain are known in the art (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest , Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). The light chain constant region can be a kappa or lambda constant region.

In order to generate the scFv nucleotide sequence, a fragment of the nucleotide sequence encoding the variable region of the heavy chain and the variable region of the light chain is operably linked to another fragment encoding a linker, for example, encoding (Gly-Ser) _n residues The linker fragment connects the nucleotide sequence of the heavy chain variable region and the light chain variable region so that the nucleotide sequence shows a continuous single-chain protein (including the heavy chain variable region, light chain variable region and Linker) (see, for example, Bird et al., 1988 Science 242: 423-426, Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883, McCafferty et al., (1990) Nature 348 :552-554).

In some embodiments, any of the nucleic acids described herein can be integrated into a performance vector to construct a vector that can encode an anti-Mia antibody, and transfect the vector into a host cell. Generally speaking, the nucleotide sequence integrated into the vector does not include introns. In this embodiment, it can be performed by methods known to those skilled in the art such as, but not limited to, electroporation, transfection, or transduction to transfer the vector to host cells, so that the host cells express anti-Mia antibodies. In this embodiment, the host cell may be a prokaryotic cell or a eukaryotic cell, including but not limited to bacteria, yeast, bacteriophage, mammalian cells (such as but not limited to CHO, COS, 239F, SF9 and/or other Cell line). The above method is described in C. Toyoda et al. (2014) British Blood Transfusion Society, the content of which is incorporated herein by reference. In some embodiments, when cultured in a culture medium, the host cell transiently or stably expresses the nucleic acid on the vector, thus providing a method for producing the aforementioned antibody or fragment of the present invention.

Establish a fusion tumor cell line to express resistance Mia Antibody

In an embodiment of the present invention, blood (hereinafter referred to as "377T") of a donor (numbered 377T) containing Mia antigen is collected to establish a fusion tumor cell line to express anti-Mia antibodies.

Antibody activity test through hemagglutination test

The anti-Mia antibody produced using the aforementioned methods (including vector or fusion tumor technology) will be tested for hemagglutination. In this example, the standard product (donor plasma with multispecific anti-Mia antibodies) and 1022 donor blood samples were taken from the Taiwan Blood Foundation. The standard product was identified as having multispecific anti-Mia antibodies by the previous method. Of donor plasma. In this example, the standard and anti-Mia antibody (377T) were used to detect 1022 donor blood samples, and the automatic blood grouping system (PK7300, Beckman Coulter, Tokyo, Japan) was used for interpretation and automatically distinguished hemagglutination. The test results of the standard product and the anti-Mia antibody of the present invention for samples with no hemagglutination (determined as positive for Mia antigen, that is, with Mia antigen) and samples without hemagglutination (determined as negative for Mia antigen, that is, no Mia antigen) 1 shown.

Table 1: Test results of Mia antigen for 1022 samples. The numbers in the table represent the number of samples. Anti- Mia antibody ( 377T ) standard Mia positive Mia negative Mia positive 50 1 ^a Mia negative 1 ^b 971 ^a represents that the standard product is positive for Mia and the anti-Mia antibody (377T) is negative for Mia; ^b represents the standard product is negative for Mia and the anti-Mia antibody (377T) is positive for Mia.

It can be seen from Table 1 that compared with the results of the standard test, in 1022 samples, only 2 samples have different interpretation results. The two samples were further sequenced. According to the sequencing results, it was found that the standard product was Mia positive, and the anti-Mia antibody (377T) was Mia negative (sample a), and its genotype was GYPB/GYPB (ie Normal glycoprotein B without Mia antigen, therefore, the test result of sample a should be negative, and the anti-Mia antibody (377T) test is negative as the correct result. In addition, the standard product test is Mia negative, anti-Mia antibody ( 377T) The sample that tested positive for Mia (sample b for short), its genotype was GYP.Mur/GYPB (that is, the third type of Mia blood group antigen with Mia antigen, therefore, the test result of sample b should be positive. A positive Mia antibody (377T) test is a correct result. From the above results, we can see that the anti-Mia antibody (377T) has better sensitivity and specificity than the standard product.

anti- Mia Antibody( 377T ) Amino acid sequence analysis

In this experimental example, the sequence analysis of the anti-Mia antibody revealed that the anti-Mia antibody includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes CDR-H1 with the amino acid sequence of SEQ ID NO. 7 (GYTFTNYAIH), CDR-H2 with the amino acid sequence of SEQ ID NO. 8 (WINAGNGNTKYSQKFQG), and CDR-H2 with the amino acid sequence of SEQ ID NO. 9 (ARDFRIPSLAVAEVGLDY). ) CDR-H3 of the amino acid sequence. The light chain variable region includes CDR-L1 having the amino acid sequence of SEQ ID NO.10 (KSSQSVLYSSNNKNYLA), CDR-L2 having the amino acid sequence of SEQ ID NO.11 (WASTRES), and CDR-L2 having the amino acid sequence of SEQ ID NO.12 (QQLYSTPLT ) CDR-L3 of the amino acid sequence. In this embodiment, any method known in the art for analyzing the amino acid sequence of an antibody can be used to perform the amino acid sequence analysis of the anti-Mia antibody (377T).

anti- Mia Antibody( 377T ) Antigen binding epitope analysis ( epitope analysis )

Next, the antigen binding epitope analysis was performed to identify the amino acid sequence of the Mia epitope bound to the anti-Mia antibody (377T). Please refer to Figure 1. In this experimental example, SEQ ID NO. 1 to SEQ ID NO. 6, SEQ ID NO. 17 to SEQ ID NO. 18 were synthesized for epitope analysis. The amino acid sequences shown on the horizontal axis of Fig. 1 from left to right are SEQ ID NO. 17, SEQ ID NO. 1 to SEQ ID NO. 6, and SEQ ID NO. 18, respectively. The peptide synthesized by the above amino acid sequence was prepared in DMSO and used for ELISA analysis. Add a 96-well plate to 50 uL per well of phosphate buffered saline (PBS) containing 20 ug/ml antigen (ie the peptide of the above amino acid sequence), and coat at 4°C until the next day. After removing the phosphate buffer, add 300 uL of barrier buffer (containing 3% skimmed milk) to each well and incubate at 37°C for 1 hour. After removing the blocking buffer, add 50 uL to each well with anti-Mia antibody (377T), and incubate at 37°C for 1 hour. After removing the anti-Mia antibody (377T) and washing with washing solution, add phosphate buffer containing anti-human secondary antibody (Leadgene human IgG-HRP, concentration 1:5000 + Leadgene human IgM-HRP, concentration 1:5000) Solution T (containing Tween 20, PBST), incubate at 37°C for 1 hour. After removing the unbound anti-human secondary antibody and washing with washing solution, add 50 uL of matrix (Leadgene commercial TMB) to each well and incubate at 37°C for 10 minutes. Then add 50 uL of stop solution (containing 0.2 MH ₂ SO ₄ ) to each well and detect the absorbance at 450 nm with a luminometer. As shown in the figure, the anti-Mia antibody (377T) and the peptide consisting of the amino acid sequence of SEQ ID NO.1 to SEQ ID NO.6 have high absorbance at 450 nm, indicating that the anti-Mia antibody (377T) and the peptide The epitope composed of these amino acid sequences has high binding power.

In summary, the anti-Mia antibody of the present invention can specifically bind to the epitope of the Mia antigen, and has higher sensitivity and specificity than the standard product. The anti-Mia antibody, kit and method produced by the present invention can detect whether the Mia antigen is contained in the sample of the organism, to confirm whether the sample is suitable for the purpose of blood transfusion, for the institution that needs a large amount of blood samples such as blood bank or hospital , Provide convenient and accurate test methods, kits and antibodies.

The above description is only illustrative, and not restrictive. Any equivalent modifications or alterations that do not depart from the spirit and scope of the present invention should be included in the scope of the attached patent application.

Figure 1 shows the results of the binding epitope analysis of the antibody or its fragments of the present invention and the Yadapu blood group antigen.

Claims (15)

An isolated antibody or fragment thereof that selectively binds specifically to an epitope of the Miltenberger blood group antigen, wherein the epitope is selected from SEQ ID NO. 1 to SEQ ID NO. Part of the amino acid sequence in the group consisting of 6 and at least contains the amino acid sequence Arg-Asp-Thr-Tyr. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment includes: A heavy chain variable region, including CDR-H1 with the amino acid sequence of SEQ ID NO.7, CDR-H2 with the amino acid sequence of SEQ ID NO.8 and CDR-H2 with the amino acid sequence of SEQ ID NO.9 H3; and A light chain variable region, including CDR-L1 with the amino acid sequence of SEQ ID NO. 10, CDR-L2 with the amino acid sequence of SEQ ID NO. 11, and CDR with the amino acid sequence of SEQ ID NO. 12 -L3. An isolated antibody or fragment thereof has a binding domain, the binding domain comprising: A heavy chain variable region, including CDR-H1 with the amino acid sequence of SEQ ID NO.7, CDR-H2 with the amino acid sequence of SEQ ID NO.8 and CDR-H2 with the amino acid sequence of SEQ ID NO.9 H3; and A light chain variable region, including CDR-L1 with the amino acid sequence of SEQ ID NO. 10, CDR-L2 with the amino acid sequence of SEQ ID NO. 11, and CDR with the amino acid sequence of SEQ ID NO. 12 -L3. The antibody or fragment thereof according to item 2 or 3 of the scope of patent application, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO.13. The antibody or fragment thereof according to item 2 or 3 of the scope of patent application, wherein the light chain variable region has the amino acid sequence of SEQ ID NO.14. The antibody or fragment thereof according to claim 4, wherein the heavy chain variable region is encoded by the nucleotide sequence of SEQ ID NO.15. The antibody or fragment thereof according to item 5 of the patent application, wherein the light chain variable region is encoded by the nucleotide sequence of SEQ ID NO.16. The antibody or fragment thereof according to any one of items 1 to 3 of the scope of patent application, wherein the antibody or fragment thereof is selected from the group consisting of monoclonal antibodies, humanized antibodies, recombinant antibodies, human antibodies, and antibodies A group consisting of biologically active fragments, antibody mimics and any combination thereof. The antibody or fragment thereof according to any one of items 1 to 3 in the scope of patent application, wherein the antibody or fragment thereof is selected from Fab, Fab', F(ab') ₂ , Fv fragments, scFv antibodies Fragments, linear antibodies, single domain antibodies, and any combination thereof. A kit for detecting a Miltenberger blood group antigen in a sample of a life object, the kit including the antibody or the antibody described in any one of items 1 to 9 of the scope of patent application Fragment. The kit according to claim 10, wherein the sample is whole blood or isolated red blood cells. The kit according to item 10 of the scope of patent application, wherein the biological system is human. A method for detecting a Miltenberger blood group antigen in a lifetime object, the method includes: Prepare a sample of the organism; and Add the antibody or fragment thereof as described in any one of items 1 to 9 of the scope of the patent application to the sample to detect whether the sample contains the Miltenberger blood group antigen. The method described in item 13 of the scope of patent application, wherein the sample is whole blood or isolated red blood cells. The method described in item 13 of the scope of patent application, wherein the biological system is human.

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