Anti-CD235a monoclonal antibody and application thereof

Abstract

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

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an anti-CD235a monoclonal antibody and an application thereof. Background Art

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

[0003] Anti-CD235a antibodies can be used in a variety of research and clinical applications. For example, they can be used to detect and study erythropoiesis, diagnose erythroleukemia, and detect a variety of hematological diseases. In addition, anti-CD235a antibodies can also be used to prepare rapid whole blood agglutination reagents, which are very important for blood transfusion and cross-matching.

[0004] In some cases, anti-CD235a antibodies may be associated with the invasion of red blood cells by Plasmodium falciparum, indicating its potential role in immune responses. In addition, anti-CD235a antibodies may also bind to tumor-associated glycogen, 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 of the invention

[0006] The present invention aims to solve at least one of the technical problems in the related art to a certain extent. The present invention provides an anti-CD235a monoclonal antibody and its application. The provided anti-CD235a monoclonal antibody can be applied to the detection of biomarkers on red blood cells, so as to be used to diagnose diseases related to proteins on red blood cells or erythroid cells as biomarkers or to be used in a variety of experimental techniques, such as flow cytometry, immunofluorescence, immunohistochemistry, western blot, etc.

[0007] The first aspect of the present invention 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 includes HCDR1 to HCDR3 sequences shown in SEQ ID NOs: 1, 2 and 3, or sequences having one or two conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOs: 1, 2 and 3;

[0009] The light chain variable region includes LCDR1-LCDR3 sequences shown in SEQ ID NOs: 4, 5 and 6, or sequences having one or two conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOs: 4, 5 and 6.

[0010] According to an embodiment of the present invention, the heavy chain variable region includes HCDR1-HCDR3 sequences shown in SEQ ID NOs: 1, 2 and 3; the light chain variable region includes LCDR1-LCDR3 sequences shown in SEQ ID NOs: 4, 5 and 6. According to an embodiment of the present invention, the sequences shown in SEQ ID NOs: 1-6 are obtained according to the Kabat database definition scheme.

[0011] The second aspect of the present invention provides an anti-CD235a monoclonal antibody or an antigen-binding fragment thereof, comprising 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.

[0012] According to an embodiment of the present invention, 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 with 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 with the sequence shown in SEQ ID NO:8.

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

[0015] According to an embodiment of the present invention, the heavy chain sequence of the anti-CD235a monoclonal antibody or the 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 invention provides a polynucleotide encoding the above-mentioned anti-CD235a monoclonal antibody or antigen-binding fragment thereof.

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

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

[0019] A sixth aspect of the present invention provides a pharmaceutical composition comprising:

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

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

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

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

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

[0025] The ninth aspect of the present invention provides a method for detecting the expression of CD235a protein in a sample, characterized by comprising:

[0026] Incubating the anti-CD235a monoclonal antibody or antigen-binding fragment described in any embodiment of the first aspect with the sample;

[0027] The incubated samples are tested to determine the expression of CD235a protein in the samples.

[0028] The sample mentioned can be an in vitro sample. The sample mentioned can be a blood sample or other sample including red blood cells or red blood cells. The detection method mentioned is used for non-disease diagnosis purposes.

[0029] The tenth aspect of the present invention provides use of the above-mentioned anti-CD235a monoclonal antibody or antigen-binding fragment thereof in the preparation of a drug or a kit.

[0030] According to an embodiment of the present invention, the drug is used for the diagnosis of diseases related to which proteins on red blood cells are used as biomarkers.

[0031] According to an embodiment of the present invention, the kit is used to detect biomarkers on red blood cells or erythroid cells.

[0032] The beneficial effects achieved by the present invention are:

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

[0034] Figure 1 It is the ELISA test result of the antibody provided according to the embodiment of the present invention.

[0035] Figure 2 It is the flow cytometry detection result of different antibodies provided according to the embodiments of the present invention.

[0036] Figure 3 This is the result of gating red blood cells by flow cytometry using the anti-CD235a monoclonal antibody provided in an embodiment of the present invention.

[0037] Figure 4 It is a flow cytometry detection diagram of different antibodies provided according to the embodiments of the present invention. DETAILED DESCRIPTION

[0038] Embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

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

[0040] Herein, the term "antibody" is used in the broadest sense, referring to a protein or polypeptide comprising an antigen binding site or antigen binding fragment or antigen binding portion, covering natural antibodies and artificial antibodies of various structures, including but not limited to complete antibody forms or antigen binding fragments of antibodies. According to a specific embodiment, the antibody mentioned is a monoclonal antibody. The so-called "complete antibody" is a protein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (abbreviated as CH). The heavy chain constant region includes a heavy chain constant domain CH1, a heavy chain constant domain CH2, and a heavy chain constant domain CH3. Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant domain CL. The VH and VL regions can be further divided into complementary determining regions (also called hypervariable regions or hypervariable regions, abbreviated as CDR), which are separated by a conserved framework region (FR). Each VH and VL contains three CDRs and four FRs, arranged from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus) in the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The CDRs of the heavy chain variable region are called HCDR1, HCDR2 and HCDR3 respectively from the amino terminus, and the CDRs of the light chain variable region are called LCDR1, LCDR2 and LCDR3 respectively from the amino terminus.

[0041] "Antigen binding fragment" refers to a portion of a full-length antibody that exhibits the property or function of binding to an antigen. The term "variable region" or "variable domain" refers to the domain involved in the binding of an antigen to an antibody heavy chain or light chain. As mentioned above, the hypervariable or hypervariable regions of the heavy and light chains of a complete antibody exhibit specific binding to an 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, and the like. Papain digestion of a complete antibody produces two identical antigen binding fragments, called Fab fragments, each of which contains a heavy and light chain variable region and a light chain constant domain and a heavy chain constant domain CH1. The Fab' fragment differs from the Fab fragment by having a few additional residues at the carboxyl terminus of the heavy chain constant domain CH1, including one or more cysteines from the hinge region of the antibody. Pepsin digestion of a complete antibody yields a F(ab')2 fragment. The F(ab')2 fragment has two antigen-binding F(ab) parts linked together by a disulfide bond, and the F(ab')2 fragment is a bivalent antibody. A single-chain antibody is a fusion protein formed by connecting the variable region of the heavy chain and the variable region of the light chain of an antibody through a flexible short peptide consisting of about 10-25 amino acids. A single-domain antibody is an antibody fragment consisting of the variable region of a single monomer. Since single-domain antibodies are usually derived from the variable region of the heavy chain of camelid antibodies or shark antibodies, they are often called nanobodies. Nanobodies contain only the heavy chain CDR region and are the smallest antigen-binding fragment with complete functions. Nanobodies include three CDR regions (CDR1-CDR3) and four framework regions FR (FR1-FR4). Framework regions FR1, FR2, FR3 and FR4 are separated by complementary determining regions CDR1, CDR2 and CDR3, respectively. Antigen-binding fragments can be obtained by recombinant means. For example, a nucleic acid encoding a target antigen-binding fragment can be constructed, introduced into an expression vector, and expressed in a suitable host to produce an antigen-binding fragment.

[0042] The term "comprising" or "including" means including the mentioned elements or steps, but not excluding other elements or steps. Of course, unless otherwise stated, comprising or including also covers the situation consisting of the mentioned elements or steps. For example, when referring to an antibody variable region comprising a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

[0043] The "affinity" or "binding affinity" mentioned herein is understood according to the common meaning in the art, and is used to reflect the strength and / or stability of the binding sites between an antigen and an antibody or an antigen-binding fragment.

[0044] "Specific binding" or "specifically binds to", "binds to", "specifically targets" a specific antigen or epitope, or "has specificity" or "can bind to" a specific antigen or epitope means to distinguish it from non-specific interactions, and this specific binding can be measured by some 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. For example, cells carrying antigens can be detected by flow cytometry, and the competitive binding between the antibody to be tested and the labeled antibody can be detected by measuring the positive rate index of the cells. Since the spatial structure of the antigen on the cell surface is closer to the form existing in the body, this method can better reflect the real situation.

[0045] Whether provided herein, antibody or antigen binding fragment, or polynucleotide, is usually separable or recombinant. "Separable" refers to being able to identify and separate and / or recover from cells or cell cultures expressing polypeptides or proteins. Usually, the separated polypeptide will be prepared by at least one purification step. "Separated antibody" refers to being substantially free of other antibodies or their antigen binding fragments having different antigen specificities. "Recombinant" means that antibodies can be produced in exogenous host cells using genetic recombination techniques.

[0046] The CDR sequences of the antibodies shown herein can be obtained by combining existing database analysis. The CDR sequences of the antibodies provided are obtained by combining the Kabat definition scheme (for example, see USDept. of Health and Human Servies, "Sequences of Proteins of Immunological Interest" (1983)). It can also be obtained by definition schemes such as 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 (for example, see J. Mol. Biol. 196: 901-917 (1987)). It is known to those skilled in the art that the differences in CDRs caused by differences in definition methods are also included in the scope of protection of the present invention.

[0047] Antibodies or antigen-binding fragments

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

[0049] The heavy chain variable region includes HCDR1 to HCDR3 sequences shown in SEQ ID NOs: 1, 2 and 3, or sequences having one or two conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOs: 1, 2 and 3;

[0050] The light chain variable region includes LCDR1-LCDR3 sequences shown in SEQ ID NOs: 4, 5 and 6, or sequences having one or two conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOs: 4, 5 and 6.

[0051] Herein, "conservative amino acid mutation" means that the amino acid mutation does not cause a significant change in the conformational structure of the protein or polypeptide, and retains its biological activity. The conservative amino acid mutation mentioned can be a mutation between different amino acids with similar physiological and biochemical properties. For example, after conservative amino acid mutation, the provided antibody still retains specific binding activity with the CD235a protein. According to an embodiment, the conservative amino acid mutation mentioned can be a substitution, deletion or increase of conservative amino acids. According to a specific embodiment, the deletion of conservative amino acids is based on the listed CDR sequences, and one amino acid, two amino acids or three amino acids are deleted. According to a specific embodiment, the increase of conservative amino acids is based on the listed CDR sequences, and one amino acid, two amino acids or three amino acids are added. According to a specific embodiment, "conservative amino acid substitution" means replacing another amino acid residue with a different amino acid residue having a side chain with similar physiological and chemical properties. For example, conservative amino acid substitutions may occur between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between residues with acidic side chains (e.g., Asp, Glu), between amino acids with basic side chains (e.g., His, Lys, and Arg), or between residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative amino acid substitutions generally do not cause significant changes in the conformational structure of the protein, and thus the biological activity of the protein can be retained. The conservative amino acid substitutions mentioned may be 1 conservative amino acid substitution, 2 conservative amino acid substitutions, 3 conservative amino acid substitutions, and the like. The names of the amino acids used herein are represented by standard single-letter or three-letter codes commonly used in the art. In some embodiments, the HCDR sequence provided has a conservative amino acid substitution with the HCDR sequence shown. In some embodiments, the HCDR sequences provided have a conservative amino acid deletion from the HCDR sequences shown. In some embodiments, the HCDR sequences provided have a conservative amino acid addition from the HCDR sequences shown. In some embodiments, the LCDR sequences provided have a conservative amino acid substitution from the LCDR sequences shown. In some embodiments, the LCDR sequences provided have a conservative amino acid deletion from the LCDR sequences shown. In some embodiments, the LCDR sequences provided have a conservative amino acid addition from the LCDR sequences shown.

[0052] According to a specific embodiment, the heavy chain variable region includes the HCDR1-HCDR3 sequences shown in SEQ ID NOs: 1, 2 and 3; the light chain variable region includes the LCDR1-LCDR3 sequences shown in SEQ ID NOs: 4, 5 and 6.

[0053] The present invention also provides an anti-CD235a monoclonal antibody or an antigen-binding fragment thereof, comprising HCDR1, HCDR2 and HCDR3 sequences of the heavy chain variable region as shown in SEQ ID NO: 7, and LCDR1, LCDR2 and LCDR3 sequences of the light chain variable region as shown 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 HCDR1 to HCDR3 sequences can be obtained by combining different definition schemes with the heavy chain variable region shown in SEQ ID NO: 7, and LCDR1 to LCDR3 sequences can be obtained by combining different definition schemes with the light chain variable region shown in SEQ ID NO: 8.

[0054] According to a specific embodiment, 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 with the sequence shown 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 shown in SEQ ID NO:8.

[0056] The "sequence identity", "sequence homology" and "sequence similarity" mentioned herein refer to the degree of sequence identity when two polypeptides, proteins or nucleotide sequences are compared. In order to determine the ratio of sequence identity, it can be achieved in a variety of ways known in the art. For example, it can be obtained using public software such as BLAST, ALIGN, BLAST-2 and other software. In some specific embodiments, the sequence identity is due to conservative amino acid mutations. According to a specific embodiment, the sequence having sequence identity with the above-mentioned heavy chain variable region or light chain variable region may 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 a specific embodiment, the sequence having sequence identity with the above-mentioned heavy chain variable region or light chain variable region differs in the framework region.

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

[0058]

[0059]

[0060] Polynucleotide

[0061] The present invention also provides a polynucleotide encoding the anti-CD235a monoclonal antibody or its antigen-binding fragment described above. The polynucleotide mentioned is separable, including but not limited to DNA, RNA or cDNA, etc. The isolated polynucleotide sequence can be obtained by conventional methods in the art to encode the monoclonal antibody or antibody or antigen-binding fragment mentioned above.

[0062] Constructs

[0063] In order to prepare the antibodies mentioned in the present invention, 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 invention also provides a construct comprising the polynucleotide described above. A variety of 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 a construct. The construct can contain a variety of operating factors such as promoters, terminators, marker genes, etc. as needed, and these operating factors can be operably connected to the polynucleotide. The promoter is usually used to provide a signal to start transcription. The promoter can select lactose promoter (Lac), Trp promoter, Tac promoter, phage PL and PR promoter as needed; the terminator provides a signal for transcription termination during the transcription process, and the marker gene on the construct is often used for screening. Of course, there can also be enhancers as needed to enhance protein expression. The expression vector is not particularly limited, and can be some commercially available expression vectors, or it can be an expression vector artificially modified as needed, such as a plasmid, a phage, a virus, etc. The virus can be a plant cell virus, a mammalian cell virus, etc. The construct can express antibodies or proteins in vitro or be introduced into cells to express antibodies or proteins.

[0064] Host cells

[0065] The present invention also provides a host cell containing the above-mentioned polynucleotide or the above-mentioned construct. Any cell suitable for expressing antibodies or proteins with polynucleotides or constructs 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, insect cells of Drosophila S2 or Sf9. Host cells containing polynucleotides or constructs can be obtained by methods commonly used in the art, such as microinjection, electroporation, chemical transfection, virus-mediated transformation, etc.

[0066] Pharmaceutical composition

[0067] The present invention also provides a pharmaceutical composition, comprising: the above-mentioned antibody or antigen-binding fragment, and a pharmaceutically acceptable carrier. The prepared pharmaceutical composition can be used to diagnose a variety of diseases, and these diseases can use proteins on red blood cells as biomarkers. For example, it can be used for the diagnosis and identification of erythroleukemia, because most tumorous nucleated red blood cells in erythroleukemia express CD235a, while acute myeloid leukemia and acute lymphoblastic leukemia hardly express CD235a. For another example, it can be used for the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH), etc.

[0068] The pharmaceutical composition also includes a pharmaceutically acceptable salt, such as an acid addition salt or a base addition salt (e.g., as described in Berge, S. M. et al (1977) J. Pharma. Sci. 66: 1-19). The pharmaceutically acceptable carrier mentioned is acceptable to the subject at the dose or concentration used. 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 hexamethonium chloride, benzalkonium chloride, benzethonium chloride; amino acids such as histidine, histidine hydrochloride, glycine, glutamine, asparagine, arginine or lysine, etc. Sterile pharmaceutical preparations can be obtained by methods commonly used in the art, such as by filtering with a sterile filter membrane. Those skilled in the art can select different pharmaceutically acceptable carriers for the pharmaceutical composition as needed to prepare different dosage forms, such as freeze-dried 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, dermal, intramuscular, peritoneal, subcutaneous, nasal, oral, rectal, topical, inhalation, transdermal, etc.

[0069] Reagent test kit

[0070] The present invention also provides a kit, which includes the above-mentioned monoclonal antibody or antigen-binding fragment. The antibody or antigen-binding fragment can be used as a reagent. For example, it can be prepared into a kit for red blood cell detection. The kit can also include containers, buffer reagents, etc. as needed. These kits can be used in a variety of experimental techniques, including but not limited to flow cytometry, immunofluorescence, immunohistochemistry, and Western blot. For example, the provided antibody or antigen-binding fragment can be used in combination with flow cytometry to quickly and accurately identify the red blood cell population. It can be applied to a variety of scenarios, such as blood samples (for example, there are multiple cell types in the blood sample, including red blood cells, red blood cells or platelets, etc.), and the anti-CD235a monoclonal antibody provided by the present invention 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. With reference to this, PNH (paroxysmal nocturnal hemoglobinuria) can be further determined and diagnosed. Those skilled in the art can make corresponding choices as needed. Accordingly, the kit can also include instructions for use to facilitate the operation and use of those skilled in the art.

[0071] Methods for producing antibodies

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

[0073] The antibody or antigen-binding fragment collected from the culture can be purified to obtain a substantially pure product. "Substantially pure" means that the purity of the antibody or antigen-binding fragment is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or even greater than 99.5%, 99.6%, 99.7%, or 99.8%.

[0074] The technical scheme of the present invention is described in detail below with reference to Examples. It should be noted that these examples are only used to facilitate the understanding of those skilled in the art and should not be regarded as limiting the scope of the present invention. The embodiments of the present invention are described in detail below, and the described embodiments are exemplary and are intended to be used 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 examples are methods or reagents commonly used in the art.

[0075] Example 1

[0076] Example 1 The antibody was prepared according to the following method.

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

[0078] Animal immunization: 6-8 week old Balb / c mice were immunized with the synthesized plasmid at a dose of 100ug / mouse. After three immunizations, the original Glycophorin-A protein was coated and detected, and the mouse serum titer was evaluated using Elisa. Qualified mice were selected for fusion.

[0079] Then the mice were killed, the spleen was removed aseptically, and the spleen cell suspension was prepared by grinding. The prepared SP2 / 0 cells were mixed with mouse spleen cells in a certain proportion, and PEG was added. Under the action of PEG, spleen cells can fuse with SP2 / 0 cells to form hybridoma cells. The hybridoma supernatant was screened using the Elisa method and flow cytometry method, and clones with positive experimental results were subcloned using the limiting dilution method. The supernatant after subcloning was further screened using Elisa and flow cytometry to screen out positive hybridoma cells that can produce the desired monoclonal antibodies, clone amplify them and freeze them.

[0080] The positive hybridoma cell screened out is numbered 5C7 (the antibody sequence expressed by the positive hybridoma cell is also represented by 5C7). After genetic testing, the heavy chain variable region sequence of the antibody of the cell is shown in SEQ ID NO:7, and the light chain variable region sequence is shown in SEQ ID NO:8. Correspondingly, the heavy chain sequence is shown in SEQ ID NO:9, and the light chain sequence is shown in SEQ ID NO:10.

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

[0082] Example 2

[0083] Example 2 The binding activity of the prepared 5C7 antibody was detected.

[0084] The protein level was tested according to the following methods, including:

[0085] Use Glycophprin-A protein to coat the ELISA plate, 50ng / well, incubate overnight at 4℃. Shake off the liquid in the wells, add 100ul 1% BSA to each well, and block at 37℃ for 1h. Shake off the liquid in the wells, and add different concentrations of the test antibody at 50uL / well (the initial concentration of each antibody is 10ug / mL, 5-fold gradient dilution), and incubate at 37℃ for 45min. Wash with PBST, and add goat anti-mouse secondary antibody (1% BSA dilution, 1:10000, manufacturer: Jackson) at 50uL / well, and incubate at 37℃ for 45min. Wash with PBST, add 50uL TMB colorimetric solution for 5-10min, and then add 50ul 2M sulfuric acid to terminate the reaction. Use an ELISA reader to read the OD450nm value.

[0086] ELISA results Figure 1 As shown, the results showed that antibody 5C7 exhibited high affinity to CD235a protein, and its EC50 result was 3.263nM.

[0087] Example 3

[0088] Example 3 The prepared antibodies were further studied as follows, including:

[0089] HEK293 cells transiently expressing Glycophprin-A were used to plate cells, with a cell amount of 5×10^5 / ml*100uL, and then 50 microliters of hybridoma cell supernatant was added as a competing antibody, and 50 microliters of a commercial antibody (Biolegend, 349106) was added as a competed antibody (1:1000), and incubated at 4 degrees Celsius for 20 minutes, indicating the fluorescence color development of the commercial antibody.

[0090] The results are as follows Figure 2 HEK293 cells transiently express Glycophprin-A, and there is a positive peak when using commercial antibodies (such as Figure 2 Antibody 5C7 can compete with commercial antibodies for binding (as shown in red in the figure); Figure 2 The binding peak shifts back to the peak graph when only the diluent is added (green).

[0091] Example 4

[0092] Example 4: Using serum samples as research samples, referring to the "Chinese Expert Consensus on Flow Cytometry Detection of Paroxysmal Nocturnal Hemoglobinuria (2021 Edition)", antibody 5C7 was used to gate the red blood cell population in the serum samples. The results are as follows Figure 3 As shown. Figure 3 It can be seen that the antibody 5C7 provided by the present invention can be used to identify the red blood cell population from the serum sample.

[0093] The identified red blood cell population is then further analyzed, and the results are shown in Figure 4 As shown, the upper left figure is the result of the screened antibody 5C7, and the upper right figure is the negative control result; the lower left figure and the lower right figure correspond to the results of the purchased control products, respectively. The results show that the antibody titer provided by the present invention is significantly better than the control company's product, and can be used to help diagnose PNH disease.

[0094] In the description of this specification, the description with reference to the terms "some embodiments" or "specific implementation methods" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be construed as limitations on the present invention. Those of ordinary skill in the art can change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. An anti-CD235a monoclonal antibody or antigen-binding fragment, characterized in that: It includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1 to HCDR3 sequences shown in SEQ ID NOs: 1, 2 and 3, or sequences having one or two conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOs: 1, 2 and 3; The light chain variable region includes LCDR1 to LCDR3 sequences shown in SEQ ID NOs: 4, 5 and 6, or sequences having one or two conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOs: 4, 5 and 6; Optionally, the heavy chain variable region includes HCDR1 to HCDR3 sequences shown in SEQ ID NOs: 1, 2 and 3; The light chain variable region includes LCDR1 to LCDR3 sequences shown in SEQ ID NOs: 4, 5 and 6.

2. An anti-CD235a monoclonal antibody or antigen-binding fragment, characterized in that: The invention comprises 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.

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 to the sequence shown in SEQ ID NO:7; 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; Optionally, the sequence of the heavy chain variable region is as shown in SEQ ID NO:7, and the sequence of the light chain variable region is as shown in SEQ ID NO:8; Optionally, the heavy chain sequence of the anti-CD235a monoclonal antibody or antigen-binding fragment is as shown in SEQ ID NO:9, and the light chain sequence is as shown in SEQ ID NO:

10.

4. A polynucleotide, characterized in that The polynucleotide encodes the anti-CD235a monoclonal antibody or antigen-binding fragment of any one of claims 1 to 3; Optionally, the polynucleotide comprises the sequences shown in SEQ ID NO:11 and SEQ ID NO:

12.

5. A construct, characterized in that Comprising the polynucleotide of claim 4.

6. A host cell, characterized in that Containing the polynucleotide according to claim 4 or the construct according to claim 5.

7. A pharmaceutical composition, characterized in that Include: The anti-CD235a monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

8. A kit, characterized in that: The invention comprises the anti-CD235a monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 3.

9. A method for detecting the expression of CD235a protein in a sample, characterized in that: include: incubating the anti-CD235a monoclonal antibody or antigen-binding fragment of any one of claims 1 to 3 with the sample; Detecting the incubated sample to determine the expression of CD235a protein in the sample; Optionally, the sample comprises a blood sample or other sample comprising red blood cells or erythroid cells.

10. Use of the anti-CD235a monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 3 in the preparation of a drug or a kit; Optionally, the drug is used for the diagnosis of diseases related to which proteins on red blood cells or erythroid cells are used as biomarkers; Optionally, the kit is used to detect biomarkers on red blood cells or erythroid cells.

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