Anti-human CD13 antibody and related production method and application thereof

Abstract

The invention discloses an anti-human CD13 antibody and a related production method and application thereof, amino acid sequences of CDRL1, CDRL2 and CDRL3 in a light chain variable region of the antibody are as shown in SEQ ID NO: 1-3, amino acid sequences of CDRH1, CDRH2 and CDRH3 in a heavy chain variable region of the antibody are as shown in SEQ ID NO: 4-6, and in addition, the amino acid sequences of CDRL1, CDRL2 and CDRL3 in the light chain variable region of the antibody are as shown in SEQ ID NO: 1-3. The invention further provides an antibody conjugate, a cell sorting reagent, a detection reagent, a detection product, a pharmaceutical composition and a biological preparation, and the application prospects are wide.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an anti-human CD13 antibody and related production methods and applications thereof. Background Art

[0002] CD13 protein, also known as aminopeptidase N, is a widely expressed type II transmembrane highly glycosylated metalloproteinase that exists in the form of homodimers and has a catalytic domain facing the extracellular space. Its enzymatic products can enter the blood system to exert their effects. CD13 is mainly expressed in myeloid cells such as monocytes, macrophages, dendritic cells, and some epithelial cells and endothelial cells. It plays an important role in the maturation and activation of myeloid cells and is associated with the occurrence and development of a variety of diseases. For example, as a surface marker of tumor stem cells, it participates in the invasion and metastasis of tumors. It is also one of the important diagnostic markers for blood system diseases such as acute myeloid leukemia. It can regulate the activity of multiple cytokines and participate in antigen processing by cutting the N-terminus. It can also play a role in signal transduction, regulate receptor recycling, enhance phagocytosis, and act as a cytokine receptor independently of enzyme activity. It is a "part-time enzyme" with multiple functions.

[0003] CD13 antibodies are widely used in scientific research and medical fields. In scientific research, they are used to identify and sort cells expressing CD13 to study cell functions and disease mechanisms. In medicine, they are used as diagnostic markers to assist in the diagnosis of diseases such as leukemia. In addition, CD13 antibodies are expected to be developed as therapeutic drugs to inhibit tumor growth and metastasis by targeting CD13 on tumor cells. The application potential and market demand of CD13 antibodies make the large-scale production and preparation of CD13 antibodies particularly important. Recombinant expression technology is achieved by cloning the gene sequence encoding the antibody into a suitable expression vector and expressing it in the host cell. Commonly used expression hosts are human embryonic kidney cells (HEK293) and Chinese hamster ovary cells (CHO). HEK293 is mainly expressed by transient transfection, with a short cycle and low cost. Moreover, the host is a human cell with a low risk of exogenous factors. Integrating the antibody gene into the CHO cell genome can achieve stable expression of the antibody. Summary of the invention

[0004] In view of this, the object of the present invention is to provide an anti-human CD13 antibody and its related production method and application in the art.

[0005] Based on the current market demand for large-scale preparation of CD13 antibodies, through early large-scale phage display library screening and verification, the inventors of the present invention have obtained antibodies that can be used for cell sorting and cell phenotypic analysis, and have clarified the light chain variable region and heavy chain variable region information of the antibody through hybridoma cell sequencing technology. Phage display technology is an effective method for rapid screening of antibodies, but it is difficult to use for large-scale preparation. In order to meet the application end's demand for antibodies, the inventors of the present invention used genetic engineering technology to construct an expression plasmid capable of expressing anti-human CD13 antibodies, and used HEK293 cells as hosts to prepare CD13 monoclonal antibodies, thereby realizing large-scale preparation of the anti-human CD13 antibodies.

[0006] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0007] The first aspect of the present invention provides an anti-human CD13 antibody, wherein the amino acid sequences of CDRL1, CDRL2, and CDRL3 in the light chain variable region of the antibody are shown in SEQ ID NOs: 1-3, respectively;

[0008] The amino acid sequences of CDRH1, CDRH2 and CDRH3 in the heavy chain variable region of the antibody are shown in SEQ ID NOs: 4-6, respectively.

[0009] Further, the amino acid sequence of the light chain of the antibody is as shown in SEQ ID NO:7 or has at least 75% homology with SEQ ID NO:7;

[0010] Optionally, the amino acid sequence of the heavy chain of the antibody is as shown in SEQ ID NO:8 or has at least 75% homology to SEQ ID NO:8.

[0011] In some embodiments, the amino acid sequences corresponding to CDRL1, CDRL2, and CDRL3 described in the present invention are not limited to the amino acid sequences shown in SEQ ID NOs: 1-3, and the amino acid sequences corresponding to CDRH1, CDRH2, and CDRH3 described in the present invention are not limited to the amino acid sequences shown in SEQ ID NOs: 4-6.

[0012] In the present invention, any CDR numbering scheme (the existing CDR numbering scheme or a new CDR numbering scheme that may be generated in the future) is used to define CDR1, CDR2, and CDR3 in the light chain as shown in SEQ ID NO:7, and CDR1, CDR2, and CDR3 in the heavy chain as shown in SEQ ID NO:8, and the amino acid sequences corresponding to CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH3 (and the nucleotide sequences corresponding to the amino acid sequences) all fall within the protection scope of the present invention.

[0013] In some embodiments, the CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH3 can be defined by any one or any combination of two or more (two or more) of the IMGT numbering scheme, the Chothia numbering scheme, the Kabat numbering scheme, the Martin (enhanced Chothia) numbering scheme, the AbM numbering scheme, and the Aho numbering scheme (or other existing CDR numbering schemes or new CDR numbering schemes that may be generated in the future) to define the amino acid sequence corresponding to the light chain or heavy chain of the antibody of the present invention as described above. The sequences corresponding to CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH3 defined in the above-mentioned manner also fall within the protection scope of the present invention.

[0014] In some embodiments, the functional variants of the antibodies provided in the first aspect of the present invention are also included in the protection scope of the present invention.

[0015] In some embodiments, the functional variant refers to a protein having obvious or significant sequence homology or similarity compared to the parent antibody (the antibody described in the first aspect of the present invention), and the functional variant retains the biological activity of the parent antibody.

[0016] Functional variants encompass, for example, variants of an antibody described herein (parent antibody) that retain the ability to recognize target cells to a similar extent, to the same extent, or to a higher extent than the parent antibody. With reference to the parent antibody, functional variants may, for example, have at least about 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95% or higher homology with the parent antibody in amino acid sequence.

[0017] In some embodiments, the functional variant may, for example, comprise an amino acid sequence of a parent antibody having at least one conservative amino acid substitution. As an alternative or in addition, the functional variant may comprise an amino acid sequence of a parent antibody having at least one non-conservative amino acid substitution. In this case, the non-conservative amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant, so that the biological activity of the functional variant is improved compared to the parent antibody. Such functional variants obtained by conservative amino acid substitution or non-conservative amino acid substitution on the basis of the parent antibody also fall within the scope of protection of the present invention.

[0018] In some embodiments, conservative amino acid substitutions are known in the art and include replacing one amino acid with particular physical and / or chemical properties with another amino acid with the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be substitutions of an acidic / negatively charged polar amino acid for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a non-polar side chain for another amino acid with a non-polar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic / positively charged polar amino acid for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain for another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), an amino acid with a β-branched side chain for another amino acid with a β-branched side chain (e.g., He, Thr and Val), an amino acid with an aromatic side chain for another amino acid with an aromatic side chain (e.g., His, Phe, Trp and Tyr).

[0019] In some embodiments, the homology refers to sequence similarity with a target amino acid sequence or nucleotide sequence. Homology includes amino acid sequences with 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher homology with the amino acid sequence of the antibody provided by the present invention. Homology can be evaluated by the naked eye or computer software. When using computer software, the homology between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the homology between related sequences. The 75% or more homology can be 75%, 80%, 85%, 90% or more homology.

[0020] In some embodiments, modification of one or more amino acids in a protein generally does not affect the function of the protein. Those skilled in the art will recognize that changing a single amino acid or a small percentage of amino acids or individual additions, deletions, insertions, substitutions to an amino acid sequence are conservative modifications, wherein the changes in a protein produce proteins with similar functions. Conservative substitution tables that provide amino acids with similar functions are well known in the art. Such amino acid sequences obtained by conservative substitutions are also included in the scope of protection of the present invention.

[0021] Substitution, deletion, insertion or any combination thereof can be used to achieve the final derivative or variant. Typically, these changes are made on several amino acids to minimize the changes in the molecule, particularly the immunogenicity and specificity of the antigen-binding protein. However, larger changes can be tolerated in some cases. Amino acid substitutions are typically single bases; insertions will typically be on the order of about one to about twenty amino acid residues, although significantly larger insertions may be tolerated. The scope of deletions is about one to about twenty amino acid residues, although in some cases, deletions can be much larger.

[0022] The second aspect of the present invention provides a bispecific antibody, wherein the bispecific antibody comprises the antibody according to the first aspect of the present invention;

[0023] Optionally, the bispecific antibody further comprises a second antibody that specifically binds to another antigen.

[0024] In some embodiments, the second antibody that specifically binds to other antigens is not particularly limited, and can be other antibodies targeting CD13 or antibodies targeting any antigen other than CD13. Those skilled in the art can make routine selections based on actual needs.

[0025] The third aspect of the present invention provides a nucleic acid molecule, which encodes the antibody according to the first aspect of the present invention or the bispecific antibody according to the second aspect of the present invention.

[0026] A person skilled in the art can easily adopt known methods, such as directed evolution and point mutation, to mutate the nucleotide sequence corresponding to the antibody of the present invention. As long as the antibody of the first aspect of the present invention or the bispecific antibody of the second aspect of the present invention is encoded, it is derived from the nucleotide sequence of the present invention and is equivalent to the sequence of the present invention, it also falls within the scope of protection of the present invention.

[0027] In some embodiments, the nucleic acid molecule is isolated or purified. The sequence of the nucleic acid molecule can be obtained using conventional techniques or hybridoma technology. Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombinant methods. This is usually cloned into a vector, then transferred into a cell, and then the relevant sequence is isolated from the host cell after proliferation by conventional methods. In addition, the relevant sequence can also be synthesized by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a longer sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0028] The fourth aspect of the present invention provides an expression vector, wherein the expression vector comprises the nucleic acid molecule according to the third aspect of the present invention;

[0029] Optionally, the vector is a plasmid, a virus-derived vector, a DNA vector, an RNA vector and / or a transposon vector;

[0030] Optionally, the virus-derived vector is a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a poxvirus vector and / or a herpesvirus vector;

[0031] Optionally, the expression vector is an expression vector pCDNA3.4 whose reading frame is signal peptide-antibody light chain described in the first aspect of the present invention-T2A-antibody heavy chain described in the first aspect of the present invention;

[0032] Optionally, the signal peptide is Mouse Ig Kappa signal peptide;

[0033] Optionally, the amino acid sequence of the signal peptide is shown in SEQ ID NO:9;

[0034] Optionally, the reading frame sequence of the expression vector is shown in SEQ ID NO:10.

[0035] The fifth aspect of the present invention provides a recombinant host cell, wherein the recombinant host cell comprises the expression vector according to the fourth aspect of the present invention;

[0036] Optionally, the host cell is a mammalian cell, an insect cell and / or a yeast cell;

[0037] Optionally, the host cell is a HEK293 cell, a CHO cell, a NS0 myeloma cell, a SP2 / 0 myeloma cell, a COS cell, a BHK cell or a Vero cell.

[0038] In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the eukaryotic cell includes, but is not limited to, a mammalian cell, an insect cell, a plant cell, a yeast cell. In some embodiments, the host cell for expressing the antibody or bispecific antibody of the present invention includes Escherichia coli, a yeast cell, an insect cell, a COS cell, a CHO cell, an immune cell, etc. In some embodiments, the immune cell includes a T cell, a B cell, a NK cell, an iNKT cell, a CTL cell, a dendritic cell, a myeloid cell, a monocyte, a macrophage, or any combination thereof.

[0039] In some embodiments, the recombinant host cell is prepared by the following method: introducing the nucleic acid molecule or expression vector of the present invention as described above into the host cell, and the method of introduction includes but is not limited to: physical method, chemical method, biological method. The physical method includes but is not limited to: microinjection, electroporation, calcium phosphate precipitation, lipofection, particle bombardment; the chemical method includes but is not limited to: colloidal dispersion system, lipid-based system; the colloidal dispersion system includes but is not limited to: macromolecular complex, nanocapsule, microsphere, bead; the lipid-based system includes but is not limited to: water-in-oil emulsion, micelle, mixed micelle, liposome; the biological method includes but is not limited to: DNA vector, lentiviral vector, poxvirus vector, herpes simplex virus vector, adenovirus vector, adeno-associated virus vector.

[0040] In some embodiments, the nucleic acid molecules or expression vectors of the present invention as described above can be introduced into host cells by various suitable means, not limited to the methods listed in the present invention, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, electroporation, TALEN method, ZFN method, non-viral vector-mediated transfection (such as liposomes) or viral vector-mediated transfection (such as lentiviral infection, retroviral infection, adenoviral infection), and other physical, chemical or biological means for transfer into cells, such as transposon technology, CRISPR-Cas9 and other technologies.

[0041] A sixth aspect of the present invention provides any of the following products:

[0042] (1) an antibody conjugate, wherein the antibody conjugate is a complex formed by directly or indirectly coupling the antibody according to the first aspect of the present invention or the bispecific antibody according to the second aspect of the present invention to a detectable marker;

[0043] (2) A cell sorting reagent, wherein the cell sorting reagent is a complex formed by binding the antibody described in the first aspect of the present invention or the bispecific antibody described in the second aspect of the present invention to magnetic beads;

[0044] (3) A detection reagent, comprising the antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, or the antibody conjugate described above;

[0045] (4) A detection product, comprising the antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the antibody conjugate or the detection reagent;

[0046] (5) A pharmaceutical composition comprising the antibody according to the first aspect of the present invention or the bispecific antibody according to the second aspect of the present invention;

[0047] (6) A biological preparation comprising the pharmaceutical composition;

[0048] Optionally, the detectable marker is a bioluminescent agent, a chemiluminescent agent, an enzyme, a photosensitive diagnostic agent, a paramagnetic ion or a radionuclide;

[0049] Optionally, the detection product is a detection kit, a detection test strip or a detection chip.

[0050] In some embodiments, the bioluminescent agent includes, but is not limited to, luciferin, luciferase, and aequorin.

[0051] In some embodiments, the chemiluminescent agent includes, but is not limited to, imidazole, luminol, isoluminol, aromatic acridinium esters, acridinium salts, and oxalate esters.

[0052] In some embodiments, the enzyme includes, but is not limited to, catalase, alkaline phosphatase, urease, horseradish peroxidase, glucose oxidase, β-D-galactosidase, or glucoamylase.

[0053] In some embodiments, the photosensitive diagnostic agent includes, but is not limited to, protoporphyrin, hematoporphyrin, methylene blue, dihydroxysilicon phthalocyanine, photoporphyrin.

[0054] In some embodiments, the paramagnetic ions include, but are not limited to, iron (II), cobalt (II), chromium (III), samarium (III), ytterbium (III), manganese (II), iron (III), nickel (II), copper (II), neodymium (III), gadolinium (III), and vanadium (II).

[0055] In some embodiments, the radionuclides include, but are not limited to: 68 Ga, 86 Y. 110 In, 111 In, 177 Lu, 18 F. 52 Fe,62 Cu, 64 Cu, 11 C. 67 Cu, 94 Tc, 99m Tc, 120 I. 123 I. 124 I. 15 O.

[0056] In some embodiments, the pharmaceutical composition may further include a pharmaceutically acceptable carrier and / or excipient, which are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). The present invention has no particular limitation on the pharmaceutically acceptable carrier and / or excipient contained in the pharmaceutical composition.

[0057] In some embodiments, the dosage form of the biologic includes, but is not limited to, solutions, emulsions, suspensions, emulsions, tablets, pills, granules, capsules, suspensions, syrups, powders, sterile aqueous solutions, non-aqueous solutions, lyophilized preparations, or suppositories.

[0058] In some embodiments, the administration of the pharmaceutical composition or biologic includes, but is not limited to, subcutaneous, intradermal, intravenous, intramuscular, intrapulmonary, intraperitoneal, oral, topical, intranasal, rectal, etc. When administered orally, a coating may be formulated to protect the active ingredient in the pharmaceutical composition or biologic to prevent degradation in the stomach. In addition, the active ingredient may be administered by any device capable of being transferred to the target tissue.

[0059] In a specific embodiment, the pharmaceutical composition or biological preparation provided by the present invention can be prepared into various dosage forms according to actual needs, and the dosage that is beneficial to the patient can be determined by the clinician according to factors such as the type, age, weight and general disease condition of the subject, and the mode of administration. The mode of administration can be, for example, injection or any other suitable mode of administration known to those skilled in the art.

[0060] The seventh aspect of the present invention provides a method for producing the antibody described in the first aspect of the present invention or the bispecific antibody described in the second aspect of the present invention, the method comprising: culturing the recombinant host cell described in the fifth aspect of the present invention, and isolating the antibody or bispecific antibody from the culture product of the recombinant host cell.

[0061] In some embodiments, the present invention demonstrates through experiments that the production method can be used for large-scale preparation of the antibody, and the purity of the prepared antibody is >98%.

[0062] In a specific embodiment, the present invention first constructs an expression plasmid of a humanized anti-CD13 antibody according to the sequence of the antibody; secondly, HEK293 cells are transiently transfected to express the recombinant anti-human CD13 monoclonal antibody; and then, the recombinant anti-human CD13 monoclonal antibody with high purity is purified.

[0063] An eighth aspect of the present invention provides any of the following methods:

[0064] (1) A method for preparing the recombinant host cell according to the fifth aspect of the present invention, the method comprising: introducing the expression vector according to the fourth aspect of the present invention into a host cell to obtain the recombinant host cell according to the fifth aspect of the present invention;

[0065] (2) A method for sorting CD13 positive cells, the method comprising: using the cell sorting reagent described in the sixth aspect of the present invention to sort a cell population to be sorted;

[0066] (3) A method for detecting CD13 in a sample for non-diagnostic and non-therapeutic purposes, the method comprising: contacting the sample with the antibody of the first aspect of the present invention, the bispecific antibody of the second aspect of the present invention, the antibody conjugate, the detection reagent or the detection product of the sixth aspect of the present invention, and detecting the formation of an immune complex between CD13 protein and the antibody;

[0067] (4) A method for inhibiting CD13 activity in a sample in vitro, the method comprising: contacting the sample with the antibody described in the first aspect of the present invention or the bispecific antibody described in the second aspect of the present invention.

[0068] In some embodiments, the sample or the sample to be tested can be selected from blood, serum, plasma, urine, saliva, ascites, brain tissue, cerebrospinal fluid, non-tissue associated cells, tissue, histological preparations, etc. from the subject to be tested. The present invention has no particular limitation on the specific type of the sample or the sample to be tested, and any sample that may contain CD13 can be used as the sample or the sample to be tested.

[0069] A ninth aspect of the present invention provides any of the following applications:

[0070] (1) Use of the antibody according to the first aspect of the present invention, the bispecific antibody according to the second aspect of the present invention, the nucleic acid molecule according to the third aspect of the present invention, the expression vector according to the fourth aspect of the present invention, or the recombinant host cell according to the fifth aspect of the present invention in the preparation of an antibody conjugate for detecting CD13;

[0071] (2) Use of the antibody according to the first aspect of the present invention, the bispecific antibody according to the second aspect of the present invention, the nucleic acid molecule according to the third aspect of the present invention, the expression vector according to the fourth aspect of the present invention, or the recombinant host cell according to the fifth aspect of the present invention in the preparation of a cell sorting reagent for sorting CD13-positive cells;

[0072] (3) Use of the antibody according to the first aspect of the present invention, the bispecific antibody according to the second aspect of the present invention, the nucleic acid molecule according to the third aspect of the present invention, the expression vector according to the fourth aspect of the present invention, the recombinant host cell according to the fifth aspect of the present invention, or the antibody conjugate according to the sixth aspect of the present invention in the preparation of a detection reagent for detecting CD13;

[0073] (4) Use of the antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, the recombinant host cell described in the fifth aspect of the present invention, the antibody conjugate or the detection reagent described in the sixth aspect of the present invention in the preparation of a detection product for detecting CD13;

[0074] (5) Use of the antibody of the first aspect of the present invention, the bispecific antibody of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the expression vector of the fourth aspect of the present invention, the recombinant host cell of the fifth aspect of the present invention, the antibody conjugate, detection reagent or detection product of the sixth aspect of the present invention in detecting CD13 for non-diagnostic and non-therapeutic purposes;

[0075] (6) Use of the antibody according to the first aspect of the present invention, the bispecific antibody according to the second aspect of the present invention, the nucleic acid molecule according to the third aspect of the present invention, the expression vector according to the fourth aspect of the present invention, the recombinant host cell according to the fifth aspect of the present invention, the antibody conjugate, detection reagent or detection product according to the sixth aspect of the present invention in the preparation of a diagnostic product for diagnosing or assisting in the diagnosis of a disease with abnormal CD13 expression;

[0076] (7) Use of the antibody according to the first aspect of the present invention, the bispecific antibody according to the second aspect of the present invention, the nucleic acid molecule according to the third aspect of the present invention, the expression vector according to the fourth aspect of the present invention, or the recombinant host cell according to the fifth aspect of the present invention in the preparation of a drug for treating and / or preventing a disease with abnormal expression of CD13;

[0077] (8) Use of the antibody according to the first aspect of the present invention, the bispecific antibody according to the second aspect of the present invention, the nucleic acid molecule according to the third aspect of the present invention, the expression vector according to the fourth aspect of the present invention, the recombinant host cell according to the fifth aspect of the present invention, or the pharmaceutical composition according to the sixth aspect of the present invention in the preparation of a biological agent for treating and / or preventing a disease with abnormal CD13 expression;

[0078] Optionally, the disease with abnormal expression of CD13 is acute myeloid leukemia, liver cancer, melanoma, glioma, chronic myeloid leukemia, pancreatic cancer, colorectal cancer, kidney cancer, bladder cancer, breast cancer, rheumatoid arthritis or inflammatory bowel disease.

[0079] In some embodiments, the CD13 abnormal expression disease is a general term for a class of diseases related to CD13 abnormal expression, and is not limited to the specific disease types listed above. Any disease related to CD13 abnormal expression will fall within the protection scope of the present invention.

[0080] The present invention provides a method for diagnosing and / or assisting in diagnosing a disease with abnormal CD13 expression, the method comprising: using the antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the antibody conjugate described in the seventh aspect of the present invention, the detection reagent or the detection product to detect a test sample derived from a subject, and detecting the presence or absence or the content of CD13 protein in the test sample derived from the subject through an antigen-antibody reaction, so as to diagnose and / or assist in diagnosing whether the subject suffers from the disease with abnormal CD13 expression or is at risk of suffering from the disease with abnormal CD13 expression.

[0081] The present invention provides a method for treating and / or preventing a disease with abnormal expression of CD13, the method comprising: administering a therapeutically and / or preventively effective amount of the antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, or the pharmaceutical composition or biological preparation described in the seventh aspect of the present invention to a subject in need thereof.

[0082] In some embodiments, the subject includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, mice, amphibians and reptiles. In a specific embodiment of the present invention, the subject is preferably a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 :The results of electrophoresis of cell feed solution;

[0084] Figure 2 : Affinity capture and purification electrophoresis (Non-reduce SDS-PAGE);

[0085] Figure 3 : SEC-HPLC analysis of the purity of the purified product corresponding to the result diagram;

[0086] Figure 4 : Antibody titer test result diagram. DETAILED DESCRIPTION

[0087] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0088] The reagents and raw materials used in the present invention are easily obtained by those of ordinary skill in the art, and can be obtained from commercial sources unless otherwise specified. The experimental methods for which specific conditions are not specified in the present invention are usually carried out according to conventional conditions in the art or according to conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0089] Example 1 Expression plasmid design, construction and plasmid mass production

[0090] Construct the expression plasmid of CD13 antibody and extract the plasmid in large quantities for transient cell transfection. The specific experimental method is as follows:

[0091] 1. Expressed sequence information

[0092] The amino acid sequences corresponding to CDRL1-3 in the light chain variable region of the anti-human CD13 antibody that can be used for cell sorting and cell phenotype analysis obtained by the inventors of the present invention through preliminary research screening and verification are shown in SEQ ID NOs: 1-3, respectively, and the amino acid sequences corresponding to CDRH1-3 in the heavy chain variable region of the antibody are shown in SEQ ID NOs: 4-6, respectively, as shown in Table 1 below. It has been verified that the antibody can specifically bind to CD13 and has good specificity and affinity.

[0093] Table 1 Sequence information of anti-human CD13 antibodies

[0094]

[0095] 2. Vector construction and plasmid extraction

[0096] Using pCDNA3.4 as the expression vector and Mouse Ig Kappa signal peptide: METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 9), an expression vector with a reading frame of “antibody light chain-T2A-antibody heavy chain” was constructed and named pJD20.

[0097] The pJD20 plasmid reading frame sequence (signal peptide + light chain + T2A + heavy chain) is as follows:

[0098] (SEQ ID NO: 10). Example 2 HEK293 cell culture and antibody transient transfection expression

[0099] HEK293 was used as the host cell for recombinant expression of antibodies. The specific experimental method is as follows:

[0100] 1. Main materials

[0101] HEK293 cells (human embryonic kidney cells), stored by our company; HEK293 serum-free medium (catalog number: AS-11), Tongli Haiyuan; HEK-293 expression feed medium (catalog number: AS-18), Tongli Haiyuan; transfection reagent TA-293 (catalog number: K20001), Zhuhai Kairui; recombinant protein expression enhancer KE-293 (catalog number: K30001), Zhuhai Kairui; glutamine (catalog number: G8540-100G), Merck; expression plasmid: plasmid extracted in the above steps (pJD20). Cell shaker, Corning; cell counter, Contstar; cell culture shaker, Eppendorf S41i; large-capacity centrifuge, Hunan Xiangyi GL-21M.

[0102] 2. Experimental methods

[0103] (1) Cell culture

[0104] HEK293 cells were revived and the cell culture density was 3.5 × 10 6 / mL, and then subculture. According to the cell density, add fresh 293 serum-free medium and dilute the cell density to 0.4-0.6×10 6 / mL, culture in a cell culture shaker (37°C, CO2 concentration 5%). Requirement: The volume of the culture medium should not exceed 1 / 3 of the volume of the culture flask.

[0105] (2) Transient transfer and feeding

[0106] 1) On the day of transfection, detect cell density and viability and adjust the cell density to 3×10 6 / mL;

[0107] 2) Take 1 mg of plasmid (plasmid number: pJD20, 1055 μg / mL, 948 μL) and add it to a 50 mL centrifuge tube (tube A) pre-prepared with 20 mL of 293 serum-free medium, and gently pipette to mix; take 5 mL of TA-293 and add it to a 50 mL centrifuge tube (tube B) pre-prepared with 20 mL of 293 serum-free medium, and gently pipette to mix;

[0108] 3) Mix the liquids in tube A and tube B, mix gently, and leave at room temperature for 10 minutes to prepare the transfection reagent-plasmid complex;

[0109] 4) Use a pipette to slowly add the transfection reagent-plasmid complex to the cells at the pre-adjusted density, shaking the cell shaker flask while adding to evenly disperse the complex. After completion, move the shaker flask into a shaker and start culturing. The culture conditions are: 5% CO2, 37°C, 120rpm;

[0110] 5) After culturing for 24 h (Day 1), samples were taken to detect cell density and viability (retained samples), and 50 mL of feed medium and 6 mL of KE-293 were added in sequence, and the culture continued;

[0111] 6) Day 3, test cell density and viability (keep sample), add 50 mL feed medium, and continue culturing;

[0112] 7) On Day 4, detect cell density and viability (keep samples); on Day 5 or when the cell viability is lower than 70%, terminate the expression, transfer the feed solution into a clean centrifuge bottle, centrifuge at 8000 rpm for 20 min at room temperature, and harvest the cell supernatant for purification.

[0113] 3. Experimental results

[0114] The results of cell status monitoring during the expression process are shown in Table 2. The electrophoresis diagram of the cell feed solution is shown in Figure 1 The results showed that the antibody was successfully expressed.

[0115] Table 2 Cell status monitoring results during expression process

[0116]

[0117]

[0118] Example 3 Affinity chromatography

[0119] The recombinant anti-CD13 antibody in the cell feed solution was captured using affinity chromatography filler. The specific experimental method is as follows:

[0120] 1. Main materials

[0121] Affinity chromatography filler: Qianchun, MabPureA LX filler (5mL); chromatography system, Insect, Unique AutoPre100D.

[0122] 2. Experimental methods

[0123] (1) Column loading: Clean 5 mL of pre-loaded column and set aside for later use. Take 5 mL of MabPureA LX filler according to the correct glue-water ratio, load the lower gasket and lower plug into the pre-loaded column, pour the filler from the top and let it settle naturally. After all the filler is loaded, load the upper gasket and upper plug.

[0124] (2) Set the flow rate to 5 mL / min;

[0125] (3) Equilibration: 20 mM Tris, 100 mM NaCl, pH 7.0, 6 CV;

[0126] (4) Sample loading: Expression delivery 1L, sample loading volume 620mL;

[0127] (5) Flow through: 2508.716mAU;

[0128] (6) Reequilibration: 20 mM Tris, 100 mM NaCl, pH 7.0, 7 CV;

[0129] (7) Wash 1: 10 mM sodium citrate, 0.5 M NaCl, pH 6.0, 15 CV, 92.461 mAU-17 mL;

[0130] (8) Wash 2: 10 mM sodium citrate, pH 6.0, 6 CV;

[0131] (9) Elution: 0.2 M glycine, 0.15 M NaCl, 6% trehalose, pH 2.8, 12 CV, 1858.72 mAU-27 mL;

[0132] (10) Neutralization: The eluted sample was adjusted to pH 8.0 by adding 1 M Tris (pH 9.0) and labeled as JD20-A.

[0133] Example 4 Purification

[0134] After G25 liquid exchange and ion exchange chromatography, high-purity antibodies were obtained through purification. The specific experimental method is as follows:

[0135] 1. Main materials

[0136] Bestdex G-25 filler, Bogron; Q Bestarose FF filler, Bogron; SP Bestarose FF filler, Bogron; chromatography system, Insex, Unique AutoPre 100D.

[0137] 2. Experimental methods

[0138] (1) G25 fluid change

[0139] 1) Column treatment: 5 mL / min, water injection, 10 CV;

[0140] 2) CIP: 8 mL / min, CIP solution flushing 10 CV;

[0141] 3) Equilibration: 8 mL / min, desalting chromatography equilibration solution (20 mM PB, pH 8.0) until the baseline is stable;

[0142] 4) Sample loading: 5 mL / min, JD20-A (27 mL);

[0143] 5) Balance: 8 mL / min, collect protein peak, 852.255 mAu, volume 45 mL, marked as JD20-AG;

[0144] 6) Storage: 8 mL / min, water flushing, 10 CV; 5 mL / min, 20% ethanol flushing, 10 CV.

[0145] (2) Q purification

[0146] 1) Column loading: Clean 5 mL of the chromatography column pre-loaded with Q filler for later use, and control the flow rate during the purification process to 6 mL / min;

[0147] 2) Equilibration: 20 mM PB, pH 8.0, 10 CV;

[0148] 3) Sample loading: JD20-AG (45 mL);

[0149] 4) Solution B: 20 ​​mM PB, 1 M NaCl, pH 8.0;

[0150] 5) Elution: 20% B elution, 1223.98 mAU-39 mL, labeled as JD27-AGQ;

[0151] (3) SP purification

[0152] 1) Column loading: Clean 5 mL of the chromatography column pre-loaded with SP filler for later use, and control the flow rate during the purification process to 5 mL / min;

[0153] 2) Equilibration: 50 mM NaAC-HAC, pH 5.5, 10 CV;

[0154] 3) Sample treatment: JD20-AGQ, 39 mL, diluted with water to a conductivity of 2.14 mS / cm, adjusted to pH 5.0, final volume 135 mL;

[0155] 4) Solution B: 25mM NaAC-HAC, 1M NaCl, pH 5.0;

[0156] 5) Elution: 10% B elution, 1966.403mAU-25mL (No.: JD20-AGQS);

[0157] 6) Column treatment: flush the column with liquid B, 6CV.

[0158] 3. Experimental results

[0159] SDS-PAGE electrophoresis analysis of each step eluted components (electrophoresis results see Figure 2), the purity of the final cation (SP) 10% B elution product was >90%. The concentration of JD20-AGQS sample detected by BCA quantitative analysis was 3.28 mg / mL.

[0160] Example 5 Antibody Property Identification

[0161] The antibody purified above is the anti-human CD13 antibody described in Example 1. The purity and titer of the antibody were further tested. The specific experimental method is as follows:

[0162] 1. Main materials

[0163] Recombinant human CD13 protein (10051-H08H), Sino Biological Pharmaceuticals; phosphate buffer, Shanghai Yuanpei; GoatAnti-Human IgG Fc / HRP (ab97225), Abcam; TMB substrate colorimetric kit (CW0050S), Kangwei Century; multi-function microplate reader (INFINITE EPLEX), Tecan; high performance liquid chromatography (InfinityII 126), Agilent.

[0164] 2. Experimental methods

[0165] (1) HPLC detection of antibody purity

[0166] 1) Sample: Take the purified product JD20-AGQS and dilute it to 0.5 mg / mL with PBS buffer (pH 7.4) according to the protein quantification results of the BCA method;

[0167] 2) Test conditions:

[0168] Column: BioCore SEC- 7.8*300mm 3μm; mobile phase: 900mL of 0.1M Na2SO4+0.1MNa2HPO4 aqueous solution adjusted to pH 6.5 with phosphoric acid + 100mL of acetonitrile; detection wavelength: 210nm; column temperature: 30℃; isocratic elution; flow rate: 0.5mL / min; injection volume: 40μL; detection time: 30min;

[0169] 3) Take the test solution, inject it according to the chromatographic conditions, and record the chromatogram;

[0170] 4) Calculate the purity of the test sample by the area normalization method, and take the average of the two samples as the final test result.

[0171] (2) ELISA to detect antibody titer

[0172] Conventional ELISA detection: coated with recombinant human CD13 protein, 0.5μg / well; blocked with 5% skim milk powder; recombinant anti-human CD13 antibody (1g / L), diluted to 100mg / L, 10-fold dilution to 100ng / L, antibody diluent as negative control, incubated at 37℃ for 1h; secondary antibody is goat anti-human IgG Fc / HRP, 10000-fold dilution, 100μL per well, incubated at 37℃ for 50min; TMB color development, microplate reader reading, analysis results. Among them, the concentrations of antibodies are: 1:100mg / L; 2:10mg / L; 3:1mg / L; 4:100μg / L; 5:10μg / L; 6:1μg / L; 7:100ng / L; 8:0ng / L.

[0173] 3. Experimental results

[0174] SEC-HPLC analysis of the purity of the purified product showed that the purity of the antibody was 98.75% (see Figure 3 The antibody titer test results showed that 0.1ng / mL of recombinant CD13 antibody was still able to detect the presence of CD13 protein, and the antibody titer was >1×10 7 ( Figure 4 ), indicating that the CD13 antibody of the present invention has a higher specific binding ability and affinity to the antigen CD13.

Claims

1. An anti-human CD13 antibody, characterized in that: The amino acid sequences of CDRL1, CDRL2, and CDRL3 in the light chain variable region of the antibody are shown in SEQ ID NOs: 1-3, respectively; The amino acid sequences of CDRH1, CDRH2 and CDRH3 in the heavy chain variable region of the antibody are shown in SEQ ID NOs: 4-6, respectively.

2. The antibody according to claim 1, characterized in that The amino acid sequence of the light chain of the antibody is as shown in SEQ ID NO: 7 or has at least 75% homology to SEQ ID NO: 7; Optionally, the amino acid sequence of the heavy chain of the antibody is as shown in SEQ ID NO:8 or has at least 75% homology to SEQ ID NO:

8.

3. A bispecific antibody, characterized in that: The bispecific antibody comprises the antibody of claim 1 or 2; Optionally, the bispecific antibody further comprises a second antibody that specifically binds to another antigen.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody according to claim 1 or 2 or the bispecific antibody according to claim 3.

5. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule of claim 4; Optionally, the vector is a plasmid, a virus-derived vector, a DNA vector, an RNA vector and / or a transposon vector; Optionally, the virus-derived vector is a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a poxvirus vector and / or a herpesvirus vector; Optionally, the expression vector is an expression vector pCDNA3.4 whose reading frame is signal peptide-antibody light chain according to claim 1 or 2-T2A-antibody heavy chain according to claim 1 or 2; Optionally, the signal peptide is Mouse Ig Kappa signal peptide; Optionally, the amino acid sequence of the signal peptide is shown in SEQ ID NO:9; Optionally, the reading frame sequence of the expression vector is shown in SEQ ID NO:

10.

6. A recombinant host cell, characterized in that The recombinant host cell comprises the expression vector of claim 5; Optionally, the host cell is a mammalian cell, an insect cell and / or a yeast cell; Optionally, the host cell is a HEK293 cell, a CHO cell, a NS0 myeloma cell, a SP2 / 0 myeloma cell, a COS cell, a BHK cell or a Vero cell.

7. Any of the following products, characterized in that: The products include: (1) An antibody conjugate, wherein the antibody conjugate is a complex formed by directly or indirectly coupling the antibody according to claim 1 or 2 or the bispecific antibody according to claim 3 to a detectable marker; (2) A cell sorting reagent, characterized in that the cell sorting reagent is a complex formed by the antibody according to claim 1 or 2 or the bispecific antibody according to claim 3 bound to magnetic beads; (3) A detection reagent, comprising the antibody according to claim 1 or 2, the bispecific antibody according to claim 3, or the antibody conjugate; (4) A detection product, comprising the antibody of claim 1 or 2, the bispecific antibody of claim 3, the antibody conjugate or the detection reagent; (5) A pharmaceutical composition comprising the antibody according to claim 1 or 2 or the bispecific antibody according to claim 3; (6) A biological preparation comprising the pharmaceutical composition; Optionally, the detectable marker is a bioluminescent agent, a chemiluminescent agent, an enzyme, a photosensitive diagnostic agent, a paramagnetic ion or a radionuclide; Optionally, the detection product is a detection kit, a detection test strip or a detection chip.

8. A method for producing the antibody according to claim 1 or 2 or the bispecific antibody according to claim 3, characterized in that: The method comprises: culturing the recombinant host cell according to claim 6, and isolating the antibody or bispecific antibody from the culture product of the recombinant host cell.

9. Any of the following methods, characterized in that: The method comprises: (1) A method for preparing the recombinant host cell according to claim 6, the method comprising: introducing the expression vector according to claim 5 into a host cell to obtain the recombinant host cell according to claim 6; (2) A method for sorting CD13 positive cells, the method comprising: using the cell sorting reagent described in claim 7 to sort the cell population to be sorted; (3) A method for detecting CD13 in a sample for non-diagnostic and non-therapeutic purposes, the method comprising: contacting the sample with the antibody of claim 1 or 2, the bispecific antibody of claim 3, the antibody conjugate, detection reagent or detection product of claim 7, and detecting the formation of an immune complex between CD13 protein and the antibody; (4) A method for inhibiting CD13 activity in a sample in vitro, the method comprising: contacting the sample with the antibody according to claim 1 or 2 or the bispecific antibody according to claim 3.

10. Any of the following applications, characterized in that: The applications include: (1) Use of the antibody of claim 1 or 2, the bispecific antibody of claim 3, the nucleic acid molecule of claim 4, the expression vector of claim 5 or the recombinant host cell of claim 6 in the preparation of an antibody conjugate for detecting CD13; (2) Use of the antibody according to claim 1 or 2, the bispecific antibody according to claim 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5 or the recombinant host cell according to claim 6 in the preparation of a cell sorting reagent for sorting CD13-positive cells; (3) Use of the antibody according to claim 1 or 2, the bispecific antibody according to claim 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, the recombinant host cell according to claim 6 or the antibody conjugate according to claim 7 in the preparation of a detection reagent for detecting CD13; (4) Use of the antibody of claim 1 or 2, the bispecific antibody of claim 3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the recombinant host cell of claim 6, the antibody conjugate or the detection reagent of claim 7 in the preparation of a detection product for detecting CD13; (5) Use of the antibody of claim 1 or 2, the bispecific antibody of claim 3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the recombinant host cell of claim 6, the antibody conjugate, detection reagent or detection product of claim 7 for detecting CD13 for non-diagnostic and non-therapeutic purposes; (6) Use of the antibody of claim 1 or 2, the bispecific antibody of claim 3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the recombinant host cell of claim 6, the antibody conjugate, detection reagent or detection product of claim 7 in the preparation of a diagnostic product for diagnosing or assisting in the diagnosis of a disease with abnormal CD13 expression; (7) Use of the antibody according to claim 1 or 2, the bispecific antibody according to claim 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5 or the recombinant host cell according to claim 6 in the preparation of a drug for treating and / or preventing a disease with abnormal expression of CD13; (8) Use of the antibody of claim 1 or 2, the bispecific antibody of claim 3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the recombinant host cell of claim 6 or the pharmaceutical composition of claim 7 in the preparation of a biological agent for treating and / or preventing a disease with abnormal expression of CD13; Optionally, the disease with abnormal expression of CD13 is acute myeloid leukemia, liver cancer, melanoma, glioma, chronic myeloid leukemia, pancreatic cancer, colorectal cancer, kidney cancer, bladder cancer, breast cancer, rheumatoid arthritis or inflammatory bowel disease.

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