Anti-cd235a monoclonal antibodies and uses thereof

Abstract

The application provides an anti-CD235a monoclonal antibody and application thereof. The provided monoclonal antibody can specifically bind to CD235a protein. The provided anti-CD235a monoclonal antibody has high specificity and affinity to CD235a protein, and can be applied to detection of red blood cells or erythroid cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to an anti-CD235a monoclonal antibody and application thereof. BACKGROUND

[0002] CD235a, also known as glycophorin A (GPA), is an important transmembrane glycoprotein mainly distributed on the surface of human red blood cells, with M and N blood group antigens. This glycoprotein is composed of 131 amino acids, with its amino terminal exposed outside the red blood cells and combined with multiple oligosaccharide side chains, thereby forming a structure with a large number of negative charges on the surface of red blood cells, which helps to prevent red blood cells from aggregating with each other during circulation.

[0003] Anti-CD235a antibodies can be used for various research and clinical applications. For example, they can be used for detecting and studying erythropoiesis, diagnosing erythroleukemia, and detecting various hematological diseases. In addition, anti-CD235a antibodies can also be used for the preparation of rapid whole blood agglutination reagents, which are very important for blood transfusion and cross matching.

[0004] In some cases, anti-CD235a antibodies may be related to the invasion of malignant Plasmodium into red blood cells, indicating their potential role in immune response. In addition, anti-CD235a antibodies may also bind to glycogen associated with tumors, showing potential in disease diagnosis.

[0005] Anti-CD235a antibodies have important value in medical research and clinical applications, especially in the fields of hematology and immunology. Anti-CD235a antibodies need to be further improved. SUMMARY

[0006] The present application aims to at least partially solve one of the technical problems in the related art. The present application provides an anti-CD235a monoclonal antibody and application thereof. The provided anti-CD235a monoclonal antibody can be applied to the detection of biomarkers on red blood cells, so as to be used for the diagnosis of related diseases with red blood cells or erythroid cells as biomarkers or in various experimental techniques, such as flow cytometry, immunofluorescence, immunohistochemistry, western blot, etc.

[0007] The first aspect of the present application provides an anti-CD235a monoclonal antibody or an antigen binding fragment thereof, comprising a heavy chain variable region and a light chain variable region,

[0008] The heavy chain variable region comprises HCDR1-HCDR3 sequences shown in SEQ ID NO: 1, 2 and 3, or sequences with one or two conservative amino acid mutations in the HCDR sequences shown in SEQ ID NO: 1, 2 and 3;

[0009] The light chain variable region comprises LCDR1-LCDR3 sequences shown in SEQ ID NO: 4, 5 and 6, or sequences having one or two conservative amino acid mutations from the LCDR sequences shown in SEQ ID NO: 4, 5 and 6.

[0010] According to an embodiment of the present application, the heavy chain variable region comprises HCDR1-HCDR3 sequences shown in SEQ ID NO: 1, 2 and 3; and the light chain variable region comprises LCDR1-LCDR3 sequences shown in SEQ ID NO: 4, 5 and 6. According to an embodiment of the present application, the sequences shown in SEQ ID NO: 1-6 are obtained in the Kabat database definition scheme.

[0011] The second aspect of the present application provides an anti-CD235a monoclonal antibody or antigen binding fragment thereof, comprising HCDR1, HCDR2 and HCDR3 sequences of a heavy chain variable region shown in SEQ ID NO: 7, and LCDR1, LCDR2 and LCDR3 sequences of a light chain variable region shown in SEQ ID NO: 8.

[0012] According to an embodiment of the present application, in the above-mentioned anti-CD235a monoclonal antibody or antigen binding fragment thereof, the sequence of the heavy chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 7;

[0013] the sequence of the light chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 8.

[0014] According to an embodiment of the present application, the sequence of the heavy chain variable region is shown in SEQ ID NO: 7, and the sequence of the light chain variable region is shown in SEQ ID NO: 8.

[0015] According to an embodiment of the present application, the heavy chain sequence of the anti-CD235a monoclonal antibody or antigen binding fragment thereof is shown in SEQ ID NO: 9, and the light chain sequence is shown in SEQ ID NO: 10.

[0016] The third aspect of the present application provides a polynucleotide encoding the above-mentioned anti-CD235a monoclonal antibody or antigen binding fragment thereof.

[0017] The fourth aspect of the present application provides a construct comprising the polynucleotide of the third aspect.

[0018] The fifth aspect of the present application provides a host cell comprising the polynucleotide of the third aspect or the construct of the fourth aspect.

[0019] The sixth aspect of the present application provides a pharmaceutical composition comprising:

[0020] The anti-CD235a monoclonal antibody or antigen-binding fragment thereof as described above, and a pharmaceutically acceptable carrier.

[0021] The seventh aspect of the present application provides a kit comprising the anti-CD235a monoclonal antibody or antigen-binding fragment thereof as described above.

[0022] The eighth aspect of the present application provides a method for producing the anti-CD235a monoclonal antibody or antigen-binding fragment thereof, comprising:

[0023] culturing the host cell of the fifth aspect, and

[0024] collecting the anti-CD235a monoclonal antibody or antigen-binding fragment thereof from the culture.

[0025] The ninth aspect of the present application provides a method for detecting the expression of CD235a protein in a sample, characterized in that the method comprises:

[0026] incubating the anti-CD235a monoclonal antibody or antigen-binding fragment thereof of any one of the first aspect with the sample;

[0027] detecting the incubated sample to determine the expression of CD235a protein in the sample.

[0028] The sample can be an ex vivo sample. The sample can be a blood sample or other sample comprising red blood cells or erythroid cells. The detection method can be used for non-disease diagnostic purposes.

[0029] The tenth aspect of the present application provides the use of the anti-CD235a monoclonal antibody or antigen-binding fragment thereof as described above in the preparation of a medicament or a kit.

[0030] According to an embodiment of the present application, the medicament is used for the diagnosis of a disease related to the protein on red blood cells as a biomarker.

[0031] According to an embodiment of the present application, the kit is used for detecting the biomarker on red blood cells or erythroid cells.

[0032] The present application has the following beneficial effects:

[0033] The anti-CD235a monoclonal antibody provided by the present application can specifically bind to CD235a protein, has high affinity and specificity, and can be used for detection of red blood cells. It can also be used for diagnosis and detection of related diseases using red blood cell proteins as biomarkers. The anti-CD235a monoclonal antibody provided can be prepared into a kit or a drug according to the corresponding use. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 ELISA detection results of antibodies provided according to embodiments of the present application.

[0035] Figure 2 Flow detection results of different antibodies according to embodiments of the present application.

[0036] Figure 3 Flow gating results of red blood cells by anti-CD235a monoclonal antibodies according to embodiments of the present application.

[0037] Figure 4 Flow detection graphs of different antibodies according to embodiments of the present application. DETAILED DESCRIPTION

[0038] Embodiments of the present application are described in detail below, examples of which are shown in the accompanying drawings. The embodiments described below by reference to the accompanying drawings are exemplary and are intended to explain the present application, and cannot be understood as a limitation of the present application.

[0039] As used in the specification and claims, "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0040] As used herein, the term "antibody" is used in the broadest sense, and includes proteins or polypeptides that comprise an antigen binding site or an antigen binding fragment or an antigen binding portion, and encompasses both natural and artificial antibodies of various configurations, including but not limited to intact antibody formats or antigen binding fragments of antibodies. According to particular embodiments, the antibodies referred to are monoclonal antibodies. As commonly used, an "intact antibody" comprises at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region includes a heavy chain constant domain CHI, a heavy chain constant domain CH2 and a heavy chain constant domain CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant domain CL. The VHand VLregions can be further subdivided into complementarity determining regions (also known as hypervariable regions or hyper-variable regions, abbreviated herein as CDR), which are separated by framework regions (FR). Each VHand VLis composed of three CDRs and four FRs, arranged from amino-terminus (N-terminus) to carboxy-terminus (C-terminus) in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDRs of the heavy chain variable region are referred to as HCDR1, HCDR2, and HCDR3, respectively, starting from the amino-terminus, and the CDRs of the light chain variable region are referred to as LCDR1, LCDR2, and LCDR3, respectively, starting from the amino-terminus.

[0041] An "antigen binding fragment" refers to a portion of a full length antibody that retains the ability to bind to an antigen. The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antigen. As noted above, the variable regions of the heavy and light chains of an intact antibody exhibit specificity for the antigen. Examples of antigen binding fragments or antigen binding portions include, but are not limited to, Fab, Fab', F(ab')2, bispecific Fab', and Fv fragments (variable regions), linear antibodies, single chain antibodies, single domain antibodies, etc. Pepsin digests of an intact antibody produce two identical antigen binding fragments, called Fab fragments, each containing a complete light chain and a portion of a heavy chain. Fab' fragments differ from Fab fragments by the inclusion of a few additional residues at the carboxy terminus of the heavy chain, including one or more cysteines from the antibody hinge region. Papain digests of an intact antibody produce F(ab')2 fragments that have two antigen binding sites. F(ab')2 fragments contain two Fab pieces that are held together by two disulfide bonds. Single chain antibodies are fusion proteins of the heavy and light chain variable regions held together by a short linker of about 10-25 amino acids. Single domain antibodies are antibody fragments consisting of a single monomeric variable region. Since single domain antibodies are usually derived from the variable region of heavy chains of camelid antibodies or shark antibodies, they are also often referred to as nanobodies. Nanobodies contain only heavy chain CDR regions and are the smallest antigen binding fragments with full functionality. Nanobodies include three CDR regions (CDR1-CDR3) and four framework regions FR (FR1-FR4). Framework region FR1, framework region FR2, framework region FR3, and framework region FR4 are separated by complementarity determining region CDR1, complementarity determining region CDR2, and complementarity determining region CDR3, respectively. Antigen binding fragments can be obtained by recombinant means. For example, a nucleic acid encoding the antigen binding fragment of interest can be constructed, introduced into an expression vector, and expressed in a suitable host to produce the antigen binding fragment.

[0042] The terms "comprising" or "including" or "having" mean that includes the recited elements or steps, but do not preclude the presence or addition of other elements or steps. Of course, unless otherwise specified, comprising or including also contemplates embodiments consisting of the recited elements or steps. For example, when referring to an antibody variable region comprising a certain specific sequence, an antibody variable region consisting of the certain specific sequence is also intended.

[0043] The term "affinity" or "binding affinity" is understood in its usual sense in the art and is used to reflect the strength and / or stability of the interaction between an antigen and a binding site on an antibody or antigen binding fragment.

[0044] "Specifically binds" or "specifically binds to" or "binds" or "specifically targets" or "has specificity for" or "is capable of binding to" a particular antigen or epitope means distinguishable from non-specific interaction, and such specific binding can be measured by methods commonly used in the art. The ability of an antibody to bind to an antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art. Detection of cells bearing the antigen can be measured, for example, by flow cytometry, and the competition of the antibody being tested with the labeled antibody can be measured by determining the positive indicator of cell binding. Since the spatial structure of the antigen on the cell surface is closer to the form present in vivo, this method more closely reflects the real situation.

[0045] The antibodies or antigen-binding fragments, or polynucleotides provided herein are generally isolated or recombinant. "Isolated" means that the polypeptide or protein can be identified and separated from other cellular components, and is at least partially purified. Typically, an isolated polypeptide will be prepared by at least one purification step. An "isolated antibody" means that the antibody is substantially free of other antibodies or antigen-binding fragments thereof having different antigenic specificities. "Recombinant" means that the antibody can be produced using genetic recombination techniques in an exogenous host cell.

[0046] The CDR sequences of the antibodies shown herein can be obtained by analysis of existing databases. The CDR sequences of the antibodies provided are obtained using the Kabat definition scheme (see, e.g., U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983)). CDRs can also be obtained using the IMGT (Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38:D301-D307 (2010); Ehrenmann, F., Lefranc, M.-P. Cold Spring Harbor Protoc., 6:737-749 (2011)), Chothia (see, e.g., J. Mol. Biol. 196:901-917 (1987)), and the like. Those skilled in the art will recognize that variations in CDRs due to differences in definition are within the scope of the present application.

[0047] Antibodies or antigen-binding fragments

[0048] The present application provides an anti-CD235a monoclonal antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region,

[0049] the heavy chain variable region comprises HCDR1-HCDR3 sequences as set forth in SEQ ID NOs: 1, 2, and 3, or sequences with one or two conservative amino acid mutations from the HCDR sequences set forth in SEQ ID NOs: 1, 2, and 3;

[0050] the light chain variable region comprises LCDR1-LCDR3 sequences as set forth in SEQ ID NOs: 4, 5, and 6, or sequences with one or two conservative amino acid mutations from the LCDR sequences set forth in SEQ ID NOs: 4, 5, and 6.

[0051] As used herein, "conservative amino acid substitution" refers to a substitution of an amino acid with another amino acid that does not result in a significant change in the conformational structure of the protein or polypeptide, and retains its biological activity. The conservative amino acid substitution can be a substitution between different amino acids that have similar physiochemical properties. For example, the provided antibodies can still retain the specific binding activity to CD235a protein after the conservative amino acid substitution. According to embodiments, the conservative amino acid substitution can be a substitution, deletion, or addition of a conservative amino acid. According to embodiments, the deletion of a conservative amino acid is a deletion of one, two, or three amino acids based on the listed CDR sequences. According to embodiments, the addition of a conservative amino acid is an addition of one, two, or three amino acids based on the listed CDR sequences. According to embodiments, "conservative amino acid substitution" means the replacement of one amino acid residue by another residue with a side chain of similar physicochemical properties. For example, conservative amino acid substitutions can occur between amino acid residues that have hydrophobic side chains (e.g., Met, Ala, Val, Leu, and He), between residues that have neutral, hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gin), between residues that have acidic side chains (e.g., Asp, Glu), between residues that have basic side chains (e.g., His, Lys, and Arg), or between residues that have aromatic side chains (e.g., Trp, Tyr, and Phe). As known in the art, conservative amino acid substitutions generally do not result in a significant change in the conformational structure of the protein, and thus can retain the biological activity of the protein. The conservative amino acid substitution can be 1 conservative amino acid substitution, 2 conservative amino acid substitutions, 3 conservative amino acid substitutions, etc. As used herein, the names of the amino acids are represented by the standard one-letter or three-letter codes commonly used in the art. In some embodiments, the provided HCDR sequences have one conservative amino acid substitution from the listed HCDR sequences. In some embodiments, the provided HCDR sequences have one conservative amino acid deletion from the listed HCDR sequences. In some embodiments, the provided HCDR sequences have one conservative amino acid addition from the listed HCDR sequences. In some embodiments, the provided LCDR sequences have one conservative amino acid substitution from the listed LCDR sequences. In some embodiments, the provided LCDR sequences have one conservative amino acid deletion from the listed LCDR sequences. In some embodiments, the provided LCDR sequences have one conservative amino acid addition from the listed LCDR sequences.

[0052] According to specific embodiments, the heavy chain variable region comprises HCDR1-HCDR3 sequences as set forth in SEQ ID NOs: 1, 2 and 3; and the light chain variable region comprises LCDR1-LCDR3 sequences as set forth in SEQ ID NOs: 4, 5 and 6.

[0053] The present application also provides an anti-CD235a monoclonal antibody or antigen binding fragment thereof, comprising HCDR1, HCDR2 and HCDR3 sequences of a heavy chain variable region as set forth in SEQ ID NO: 7, and LCDR1, LCDR2 and LCDR3 sequences of a light chain variable region as set forth in SEQ ID NO: 8. The provided antibody or antigen binding fragment contains HCDR1, HCDR2 and HCDR3 sequences, and LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1-HCDR3 sequences can be obtained via the heavy chain variable region as set forth in SEQ ID NO: 7 according to different definition schemes, and the LCDR1-LCDR3 sequences can be obtained via the light chain variable region as set forth in SEQ ID NO: 8 according to different definition schemes.

[0054] According to specific embodiments, the sequence of the heavy chain variable region is a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence as set forth in SEQ ID NO: 7.

[0055] The sequence of the light chain variable region is a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence as set forth in SEQ ID NO: 8.

[0056] As used herein, "sequence identity," "sequence homology," "sequence similarity" refer to the extent to which two polypeptide or protein or nucleotide sequences are identical in sequence. To determine the percent sequence identity, one can use a variety of methods known in the art. For example, one can use publicly available software such as BLAST, ALIGN, BLAST-2, etc. In some embodiments, the sequence identity is due to conservative amino acid mutations. According to embodiments, a sequence that has sequence identity to the heavy chain variable region or the light chain variable region described above can be due to 1 conservative amino acid mutation, 2 conservative amino acid mutations, 3 conservative amino acid mutations, 4 conservative amino acid mutations, 5 conservative amino acid mutations, 6 conservative amino acid mutations, 7 conservative amino acid mutations, 8 conservative amino acid mutations, 9 conservative amino acid mutations, or 10 conservative amino acid mutations. According to embodiments, a sequence that has sequence identity to the heavy chain variable region or the light chain variable region described above differs in the framework region.

[0057] According to embodiments, the heavy chain variable region is the sequence set forth in SEQ ID NO: 7; and the light chain variable region is the sequence set forth in SEQ ID NO: 8. According to embodiments, the heavy chain sequence of the anti-CD235a monoclonal antibody is set forth in SEQ ID NO: 9, and the light chain sequence is set forth in SEQ ID NO: 10.

[0058]

[0059]

[0060] Polynucleotide

[0061] The present application also provides a polynucleotide encoding the anti-CD235a monoclonal antibody or antigen binding fragment thereof described above. The polynucleotide referred to is isolatable, including but not limited to DNA, RNA, or cDNA, etc. The isolated polynucleotide sequence encoding the monoclonal antibody or antibody or antigen binding fragment referred to can be obtained using conventional methods in the art.

[0062] Construct

[0063] To prepare the antibody mentioned in the present application, the polynucleotide sequence encoding the antibody can be inserted into a replicable expression vector and expressed in a host cell or a cell-free expression system. The present application also provides a construct comprising the polynucleotide mentioned above. Various methods commonly used in the art can be used to obtain the construct, including in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. For example, the polynucleotide can be inserted into the multiple cloning site of the expression vector to form the construct. The construct can contain various operators such as promoters, terminators, marker genes, etc. as needed, which are operably linked to the polynucleotide. The promoter is usually used to provide a signal to start transcription, and the promoter can be selected as needed, such as the lactose promoter (Lac), the Trp promoter, the Tac promoter, the PL and PR promoters of the bacteriophage; the terminator provides a signal to terminate transcription during transcription, and the marker gene on the construct is commonly used for screening. Of course, enhancers can also be added as needed to enhance protein expression. The expression vector is not particularly limited and can be a commercially available expression vector or an expression vector artificially modified as needed, such as a plasmid, a bacteriophage, a virus, etc. The virus can be a plant cell virus, a mammalian cell virus, etc. The construct can express the antibody or protein in vitro or can be introduced into a cell to express the antibody or protein.

[0064] Host cell

[0065] The present application also provides a host cell containing the polynucleotide or the construct mentioned above. Any cell suitable for polynucleotide or construct expression of the antibody or protein can be used as a host cell. The host cell can be a prokaryotic cell such as a bacterial cell, or a eukaryotic cell such as a yeast cell, a mammalian cell, etc. Commonly used host cells can be yeast cells, CHO, HEK-293 cells, COS cells, Drosophila S2 or Sf9 insect cells. The host cell containing the polynucleotide or the construct can be obtained by using methods commonly used in the art, such as microinjection, electroporation, chemical transfection, virus-mediated transformation, etc.

[0066] Pharmaceutical composition

[0067] The present application also provides a pharmaceutical composition comprising: the antibody or antigen binding fragment mentioned above, and a pharmaceutically acceptable carrier. The prepared pharmaceutical composition can be used to diagnose various diseases, which can use the protein on the red blood cell as a biomarker. For example, it can be used for the diagnosis and identification of erythroleukemia, because most of the tumor nucleated red blood cells in erythroleukemia express CD235a, while acute myeloid leukemia and acute lymphoblastic leukemia almost do not express CD235a. For another example, it can be used for the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH), etc.

[0068] The pharmaceutical composition also includes pharmaceutically acceptable salts, such as acid addition salts or base addition salts (e.g., as described in Berge, S. M. et al. (1977) J. Pharma. Sci. 66: 1-19). The pharmaceutically acceptable carriers mentioned are acceptable to the subject at the dosage or concentration employed. Pharmaceutically acceptable carriers include, but are not limited to, buffers or salts, such as disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, sodium acetate, citric acid, sodium citrate, citrate, Tris; sugars, such as trehalose, polysorbate, sucrose, mannitol; surfactants such as polysorbate; preservatives such as chlorhexidine, benzalkonium chloride, benzethonium chloride; amino acids such as histidine, histidine hydrochloride, glycine, glutamine, asparagine, arginine or lysine, etc. The sterile pharmaceutical preparation can be obtained by methods commonly used in the art, such as filtration through a sterile filter. The skilled person can select different pharmaceutically acceptable carriers for the pharmaceutical composition as needed, and prepare different dosage forms, such as lyophilized dosage forms, injections, etc. The prepared different pharmaceutical dosage forms can be formulated into any suitable administration route for administration to the subject, including but not limited to intravenous, intradermal, intramuscular, peritoneal, subcutaneous, nasal, oral, rectal, topical, inhalation, transdermal, etc.

[0069] Kit

[0070] The present application also provides a kit comprising the above monoclonal antibody or antigen binding fragment. The antibody or antigen binding fragment can be in the form of a reagent. For example, a kit for detecting red blood cells can be prepared. The kit can also include containers, buffer reagents, etc. as needed. These kits can be used in various experimental techniques, including but not limited to flow cytometry, immunofluorescence, immunohistochemistry, and Western blot, etc. For example, the provided antibody or antigen binding fragment can be used in combination with flow cytometry to quickly and accurately identify a red blood cell population. It can be suitable for various scenarios, such as a blood sample (e.g., a blood sample containing various cell types, including red blood cells, red blood cells, or platelets, etc.). The anti-CD235a monoclonal antibody provided by the present application can be used to determine the red blood cell population from the blood sample, and further analyze the red blood cells, including but not limited to detecting the content of hemoglobin in the red blood cells, detecting the size of the red blood cells, etc. In this regard, PNH (paroxysmal nocturnal hemoglobinuria) can be further determined and diagnosed. The skilled person can make appropriate selection as needed. Accordingly, the kit can also include instructions for use, to facilitate the operation and use of the skilled person.

[0071] Method for producing an antibody

[0072] The present invention also provides a method for producing the above-mentioned antibody or antigen-binding fragment, comprising: culturing the above-mentioned host cells, and collecting the antibody or antigen-binding fragment from the culture.

[0073] Antibodies or antigen-binding fragments collected from cultures can be purified to obtain substantially pure products. "Substantially pure" means that the purity of the antibody or antigen-binding fragment reaches 95%, 96%, 97%, 98%, 99%, or even 99.5%, 99.6%, 99.7%, or 99.8% or higher.

[0074] The technical solution of the present invention will be described in detail below with reference to the embodiments. It should be noted that these embodiments are only for the convenience of those skilled in the art and should not be considered as limiting the scope of protection of the present invention. The embodiments of the present invention are described in detail below. These described embodiments are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention. Unless otherwise stated, the methods or reagents used in the embodiments are methods or reagents commonly used in the art.

[0075] Example 1

[0076] In Example 1, an antibody was prepared according to the following method.

[0077] Immunogen preparation: A plasmid containing Glycophorin-A encoded nucleic acid was synthesized according to Uniport P02724.

[0078] Animal immunization: Balb / c mice aged 6-8 weeks were immunized with the synthesized plasmid at a dose of 100ug / mouse. After three immunizations, the original Glycophorin-A protein was detected by coating. The serum titer of the mice was evaluated using ELISA. Mice that passed the evaluation were selected for fusion.

[0079] Mice were then euthanized, and spleens were aseptically removed and ground to prepare a spleen cell suspension. The prepared SP2 / 0 cells were mixed with mouse spleen cells at a specific ratio, and PEG was added. Under the action of PEG, spleen cells fused with SP2 / 0 cells to form hybridoma cells. The hybridoma supernatant was screened using ELISA and flow cytometry. Clones with positive results were subcloned using limiting dilution. The subcloned supernatant was further screened using ELISA and flow cytometry to identify positive hybridoma cells capable of producing the desired monoclonal antibody. These cells were then amplified and cryopreserved.

[0080] The screened positive hybridoma cell is numbered 5C7 (the antibody sequence expressed by the positive hybridoma cell is also represented by 5C7), and the gene detection shows that the heavy chain variable region sequence of the antibody of the cell is as shown in SEQ ID NO: 7, the light chain variable region sequence is as shown in SEQ ID NO: 8, the corresponding heavy chain sequence is as shown in SEQ ID NO: 9, and the light chain sequence is as shown in SEQ ID NO: 10.

[0081] Then, the obtained antibody is expressed and purified to obtain the antibody with a purity of at least 95%, which is numbered 5C7, and the performance of the antibody is studied.

[0082] Example 2

[0083] Example 2 detects the binding activity of the prepared 5C7 antibody.

[0084] The detection is carried out at the protein level according to the following method, which includes:

[0085] Glycophprin-A protein is used to coat the enzyme-labeled plate, 50ng / well, 4℃ incubation overnight. The liquid in the well is shaken off, 100ul 1% BSA is added to each well, and the plate is sealed at 37℃ for 1h. The liquid in the well is shaken off, and different concentrations of the antibody to be tested are added according to 50uL / well (the initial concentration of each antibody is 10ug / mL, 5 times gradient dilution), and the plate is incubated at 37℃ for 45min. PBST is used for cleaning, and 50uL / well of goat anti-mouse secondary antibody (1% BSA dilution, 1:10000, manufacturer: Jackson) is added, and the plate is incubated at 37℃ for 45min. PBST is used for cleaning, 50uL of TMB color developing liquid is added for color development for 5-10min, and then 50ul of 2M sulfuric acid is added to terminate the reaction. The OD450nm value is read by an enzyme-labeled instrument.

[0086] The ELISA results are as shown in Figure 1 The results show that the antibody 5C7 exhibits high affinity with CD235a protein, and the EC50 result is 3.263nM.

[0087] Example 3

[0088] Example 3 further studies the prepared antibody, which includes:

[0089] The HEK293 cells expressing Glycophprin-A instantaneously were plated with a cell amount of 5x10^5 / ml*100uL, then 50 microliters of hybridoma cell supernatant were added as competitive antibodies, and 50 microliters of commercial antibodies (Biolegend, 349106) were added as competed antibodies (1:1000), and incubated at 4 degrees Celsius for 20 minutes, and the fluorescence coloration of the commercial antibodies was indicated.

[0090] The results are shown in Figure 2 . The HEK293 cells express Glycophprin-A instantaneously, and the positive peak exists in the presence of commercial antibodies (as shown in Figure 2 the middle red); the antibody 5C7 can compete with the commercial antibodies to bind (as shown in Figure 2 the middle blue), so that the binding peak moves back to the peak chart direction of the diluent (green) only.

[0091] Example 4

[0092] Example 4 uses serum samples as research samples, refers to the “Chinese Expert Consensus on Flow Cytometry Detection of Paroxysmal Sleep Hemoglobinuria (2021 Edition)”, and uses the antibody 5C7 to circle the red blood cell group in the serum sample, and the results are shown in Figure 3 . As can be seen from Figure 3 , the antibody 5C7 provided by the application can be used to circle the red blood cell group from the serum sample.

[0093] Then the circled red blood cell group is further analyzed, and the results are shown in Figure 4 , wherein the upper left graph is the result of the screened antibody 5C7, and the upper right graph is the negative control result; the lower left graph and the lower right graph correspond to the results of the purchased control product, respectively, and the results show that the antibody provided by the application is obviously better than the control company product, and can be used to help diagnose PNH disease.

[0094] In the description of the specification, the description of the terms “some embodiments” or “specific embodiments” means that the specific features, structures, materials or characteristics described in conjunction with the embodiments or examples are contained in at least one embodiment or example of the application. In the specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Although the embodiments of the application have been shown and described above, it is understood that the above embodiments are exemplary and cannot be understood as limiting the application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the application.

Claims

1. An anti-CD235a monoclonal antibody or antigen-binding fragment, characterized in that, Includes variable regions of heavy chains and variable regions of light chains. The heavy chain variable region includes the HCDR1 sequence shown in SEQ ID NO:1, the HCDR2 sequence shown in SEQ ID NO:2, and the HCDR3 sequence shown in SEQ ID NO:3; The light chain variable region includes the LCDR1 sequence shown in SEQ ID NO:4, the LCDR2 sequence shown in SEQ ID NO:5, and the LCDR3 sequence shown in SEQ ID NO:

6.

2. An anti-CD235a monoclonal antibody or antigen-binding fragment, characterized in that, Includes the HCDR1, HCDR2 and HCDR3 sequences of the heavy chain variable region as shown in SEQ ID NO:7, and the LCDR1, LCDR2 and LCDR3 sequences of the light chain variable region as shown in SEQ ID NO:8; The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences are obtained according to any of the Kabat, IMGT, or Chothia definition schemes.

3. The anti-CD235a monoclonal antibody or antigen-binding fragment according to claim 1 or 2, characterized in that, The sequence of the heavy chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:7; The sequence of the variable region of the light chain has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:

8.

4. The anti-CD235a monoclonal antibody or antigen-binding fragment according to claim 1 or 2, characterized in that, The sequence of the heavy chain variable region is shown in SEQ ID NO:7, and the sequence of the light chain variable region is shown in SEQ ID NO:

8.

5. The anti-CD235a monoclonal antibody or antigen-binding fragment according to claim 1 or 2, characterized in that, The heavy chain sequence of the anti-CD235a monoclonal antibody or antigen-binding fragment is shown in SEQ ID NO:9, and the light chain sequence is shown in SEQ ID NO:

10.

6. A polynucleotide, characterized in that, The polynucleotide encodes the anti-CD235a monoclonal antibody or antigen-binding fragment as described in any one of claims 1 to 5.

7. The polynucleotide according to claim 6, characterized in that, The polynucleotide includes the sequences shown in SEQ ID NO:11 and SEQ ID NO:

12.

8. A construct, characterized in that, It comprises the polynucleotide as described in claim 6 or 7.

9. A host cell, characterized in that, Contains the polynucleotide of claim 6 or 7 or the construct of claim 8.

10. A reagent kit, characterized in that, Includes the anti-CD235a monoclonal antibody or antigen-binding fragment as described in any one of claims 1 to 5.

11. A method for detecting CD235a protein expression in samples for non-disease diagnostic purposes, characterized in that, include: The sample is incubated with the anti-CD235a monoclonal antibody or antigen-binding fragment as described in any one of claims 1 to 5. The incubated samples were tested to determine the expression of CD235a protein in the samples.

12. The method according to claim 11, characterized in that, The sample includes blood samples or other samples that include red blood cells or erythroid cells.

13. Use of the anti-CD235a monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 5 in the preparation of a kit for detecting CD235a.

14. The use according to claim 13, characterized in that, The kit is used to detect biomarkers on erythrocytes or erythroid cells.

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