An antibody against human CD13, its related production methods and applications

Abstract

The present invention discloses an anti-human CD13 antibody and its related production methods and applications. The amino acid sequences of CDRL1, CDRL2, and CDRL3 in the light chain variable region of the antibody are shown in SEQ ID NO: 1-3, and the amino acid sequences of CDRH1, CDRH2, and CDRH3 in the heavy chain variable region of the antibody are shown in SEQ ID NO: 4-6. In addition, the present invention also provides antibody conjugates, cell sorting reagents, detection reagents, detection products, pharmaceutical compositions, and biological agents, which have broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to an antibody against human CD13, and its related production methods and applications. Background Art

[0002] The CD13 protein, also known as aminopeptidase N, is a widely expressed type II transmembrane highly glycosylated metalloprotease that exists in the form of a homodimer and has a catalytic domain facing the extracellular space. Its enzymatic products can enter the bloodstream to play a role. CD13 is mainly expressed in myeloid cells such as monocytes, macrophages, dendritic cells, and some epithelial cells and endothelial cells, and plays an important role in the maturation and activation of myeloid cells. It is also related to the occurrence and development of various diseases. For example, it participates in the invasion and metastasis of tumors as a surface marker of cancer stem cells and is also one of the important diagnostic markers for hematological diseases such as acute myeloid leukemia. It can regulate the activities of various cytokines by cleaving the N-terminus and participate in antigen processing. It can also play functions such as signal transduction, regulation of receptor recycling, enhancement of phagocytosis, and acting as a cytokine receptor independently of its enzymatic activity, and is a "moonlighting enzyme" with multiple functions.

[0003] CD13 antibodies are widely used in the fields of scientific research and medicine. In scientific research, they are used to identify and sort cells expressing CD13 and study cell functions and disease mechanisms. In medicine, they are used as diagnostic markers to assist in the diagnosis of diseases such as leukemia. Moreover, CD13 antibodies are expected to be developed into 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 host cells. Commonly used expression hosts include human embryonic kidney cells (HEK293) and Chinese hamster ovary cells (CHO). HEK293 is mainly used for transient transfection and expression, with a short cycle, low cost, and being a human-derived 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 purpose of the present invention is to provide an antibody against human CD13, and its related production methods and applications for this field.

[0005] Based on the current market demand for large-scale preparation of CD13 antibodies, through large-scale phage display library screening and verification in the early stage, the inventors of the present invention have obtained antibodies that can be used for cell sorting and cell phenotype analysis, and have determined 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 antibody screening, but it is difficult to be used for large-scale preparation. To meet the demand for antibodies at the application end, the inventors of the present invention constructed an expression plasmid capable of expressing anti-human CD13 antibodies using genetic engineering technology, and used HEK293 cells as the host to prepare CD13 monoclonal antibodies, realizing the large-scale preparation of the anti-human CD13 antibodies.

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

[0007] In the first aspect of the present invention, there is provided an antibody against human CD13, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 in the light chain variable region of the antibody are respectively as shown in SEQ ID NO: 1-3;

[0008] The amino acid sequences of CDRH1, CDRH2, and CDRH3 in the heavy chain variable region of the antibody are respectively as shown in SEQ ID NO: 4-6.

[0009] Furthermore, 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 with SEQ ID NO: 8.

[0011] In some embodiments, the amino acid sequences corresponding to CDRL1, CDRL2, and CDRL3 of the present invention are not limited to the amino acid sequences shown in SEQ ID NO: 1-3, and the amino acid sequences corresponding to CDRH1, CDRH2, and CDRH3 of the present invention are also not limited to the amino acid sequences shown in SEQ ID NO: 4-6.

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

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

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

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

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

[0017] In some embodiments, the functional variant may, for example, comprise the amino acid sequence of a parental antibody having at least one conservative amino acid substitution. As an alternative or in addition, the functional variant may comprise the amino acid sequence of a parental antibody having at least one non-conservative amino acid substitution. In such cases, 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 may enhance the biological activity of the functional variant such that the biological activity of the functional variant is increased compared to the parental antibody. Such functional variants obtained by conservative or non-conservative amino acid substitution based on the parental 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 substituting one amino acid having specific physical and / or chemical properties with another amino acid having the same or similar chemical or physical properties. For example, a conservative amino acid substitution may be an acidic / negatively charged polar amino acid substituting another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid having a non-polar side chain substituting another amino acid having a non-polar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic / positively charged polar amino acid substituting another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid having a polar side chain substituting another uncharged amino acid having a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), an amino acid having a β-branched side chain substituting another amino acid having a β-branched side chain (e.g., Ile, Thr, and Val), or an amino acid having an aromatic side chain substituting another amino acid having an aromatic side chain (e.g., His, Phe, Trp, and Tyr).

[0019] In some embodiments, the homology refers to the sequence similarity to a target amino acid sequence or nucleotide sequence. Homology includes amino acid sequences having 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher homology to the amino acid sequence of the antibody provided by the present invention. Homology can be evaluated by the naked eye or by 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 homology of 75% or more than 75% can be 75%, 80%, 85%, 90% or more than 95% 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, or substitutions to the amino acid sequence are conservative modifications, where the change in the protein results in a protein with similar function. Tables of conservative substitutions providing amino acids with similar function are well known in the art. The amino acid sequences obtained by such conservative substitutions are also included within the scope of the present invention.

[0021] Final derivatives or variants can be achieved using substitutions, deletions, insertions, or any combination thereof. Generally, these changes are made to a few amino acids to minimize changes to the molecule, particularly the immunogenicity and specificity of the antigen-binding protein. However, in certain cases, larger changes can be tolerated. Amino acid substitutions are typically single base; insertions will generally be on the order of about one to about twenty amino acid residues, although significantly larger insertions may be tolerated. Deletions range from 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, the bispecific antibody comprising the antibody described in 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 another antigen is not particularly limited, and it can be another antibody targeting CD13 or an antibody targeting any antigen other than CD13, which can be conventionally selected by those skilled in the art according to actual needs.

[0025] The third aspect of the present invention provides a nucleic acid molecule encoding the antibody described in the first aspect of the present invention or the bispecific antibody described in the second aspect of the present invention.

[0026] Those of ordinary skill in the art can easily mutate the nucleotide sequence corresponding to the antibody of the present invention using known methods, such as directed evolution and site-directed mutagenesis, as long as it encodes the antibody described in the first aspect of the present invention or the bispecific antibody described in the second aspect of the present invention, and is derived from the nucleotide sequence of the present invention and equivalent to the sequence of the present invention, and it also falls within the scope 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 by conventional techniques or using hybridoma techniques. Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. In addition, the relevant sequence can also be synthesized by artificial synthesis methods, especially when the fragment length is short. Usually, longer fragments can be obtained by first synthesizing multiple small fragments and then ligating them.

[0028] The fourth aspect of the present invention provides an expression vector, which contains the nucleic acid molecule described in 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 poxviral vector, and / or a herpesviral vector;

[0031] Optionally, the expression vector is the expression vector pCDNA3.4 with a reading frame of signal peptide - the antibody light chain described in the first aspect of the present invention - T2A - the 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 as shown in SEQ ID NO:9;

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

[0035] The fifth aspect of the present invention provides a recombinant host cell, which contains the expression vector described in 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, an NS0 myeloma cell, an 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 cells include, but are not limited to: mammalian cells, insect cells, plant cells, yeast cells. In some embodiments, the host cells for expressing the antibodies or bispecific antibodies of the present invention include Escherichia coli, yeast cells, insect cells, COS cells, CHO cells, immune cells, etc. In some embodiments, the immune cells include T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages, 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 as described above in the present invention into a host cell, and the introducing methods include, but are not limited to: physical methods, chemical methods, biological methods. The physical methods include, but are not limited to: microinjection, electroporation, calcium phosphate precipitation, lipofection, particle bombardment; the chemical methods include, but are not limited to: colloidal dispersion systems, lipid-based systems; the colloidal dispersion systems include, but are not limited to: macromolecular complexes, nanocapsules, microspheres, beads; the lipid-based systems include, but are not limited to: oil-in-water emulsions, micelles, mixed micelles, liposomes; the biological methods include, but are not limited to: DNA vectors, lentiviral vectors, poxviral vectors, herpes simplex virus vectors, adenoviral vectors, adeno-associated viral vectors.

[0040] In some embodiments, the nucleic acid molecule or expression vector as described above in the present invention can be introduced into a host cell 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), as well as other physical, chemical or biological means for transferring into cells, such as transposon technology, CRISPR-Cas9 and other technologies.

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

[0042] (1) An antibody conjugate, which is a complex formed by directly or indirectly conjugating 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 a detectable label;

[0043] (2) A cell sorting reagent, which 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, wherein the detection reagent comprises 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;

[0045] (4) A detection product, wherein the detection product comprises 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, wherein the pharmaceutical composition comprises the antibody described in the first aspect of the present invention or the bispecific antibody described in the second aspect of the present invention;

[0047] (6) A biological agent, wherein the biological agent comprises the pharmaceutical composition;

[0048] Optionally, the detectable label is a bioluminescent agent, chemiluminescent agent, enzyme, photosensitizing diagnostic agent, paramagnetic ion, or radionuclide;

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

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

[0051] In some embodiments, the chemiluminescent agent includes but is not limited to: imidazole, luminol, isoluminol, aromatic acridinium esters, acridinium salts, 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 photosensitizing diagnostic agent includes but is not limited to: protoporphyrin, hematoporphyrin, methylene blue, dihydroxysilylphthalocyanine, photoporphyrin.

[0054] In some embodiments, the paramagnetic ion includes but is 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), vanadium (II).

[0055] In some embodiments, the radionuclide includes but is 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 comprise a pharmaceutically acceptable carrier and / or excipient, which are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). The present invention places no particular limitation on the pharmaceutically acceptable carrier and / or excipient contained in the pharmaceutical composition.

[0057] In some embodiments, the dosage forms of the biological agent include, but are 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 methods of the pharmaceutical composition or biological agent include, but are not limited to: subcutaneous, intradermal, intravenous, intramuscular, intrapulmonary, intraperitoneal, oral, topical, intranasal, rectal, etc. When administered orally, it can be formulated into a coating that protects the active ingredient in the pharmaceutical composition or biological agent from degradation in the stomach. In addition, the active ingredient can be administered by any device capable of transferring to the target tissue.

[0059] In specific embodiments, the pharmaceutical composition or biological agent provided by the present invention can be made into various dosage forms according to actual needs, and the clinician can determine the dosage beneficial to the patient according to factors such as the type, age, weight, and general disease condition of the subject, and the administration method. The administration method can be, for example, injection or any other suitable administration method 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 experimentally demonstrates that the production method can be used for large-scale preparation of the antibody, and the purity of the prepared antibody > 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, uses HEK293 cells for transient transfection to express a recombinant anti-human CD13 monoclonal antibody; and then purifies to obtain a recombinant anti-human CD13 monoclonal antibody with high purity.

[0063] The eighth aspect of the present invention provides any one 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: sorting a cell population to be sorted using the cell sorting reagent described in the sixth aspect of the present invention;

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

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

[0068] In some embodiments, the sample or the test sample can be selected from blood, serum, plasma, urine, saliva, ascites, brain tissue, cerebrospinal fluid, non-tissue-associated cells, tissues, histological preparations, etc. derived from a test subject. The present invention does not have any particular limitation on the specific type of the sample or the test sample, and any sample that may contain CD13 can be used as the sample or the test sample.

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

[0070] (1) The application 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 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 according to the sixth aspect of the present invention, or the detection reagent in the preparation of a detection product for detecting CD13;

[0074] (5) 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 according to the sixth aspect of the present invention, the detection reagent, or the detection product in the detection of 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 according to the sixth aspect of the present invention, the detection reagent, or the detection product in the preparation of a diagnostic product for diagnosing or assisting in diagnosing diseases 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 diseases with abnormal CD13 expression;

[0077] (8) 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, or the pharmaceutical composition described in the sixth aspect of the present invention in the preparation of a biological agent for treating and / or preventing diseases with abnormal CD13 expression;

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

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

[0080] The present invention provides a method for diagnosing and / or assisting in the diagnosis of diseases with abnormal CD13 expression. The method includes: 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, detection reagent or detection product described in the seventh aspect of the present invention to detect a test sample from a subject, and detecting the presence or content of CD13 protein in the test sample from the subject through an antigen-antibody reaction, so as to diagnose and / or assist in the diagnosis of whether the subject has a disease with abnormal CD13 expression or the risk of having a disease with abnormal CD13 expression.

[0081] The present invention provides a method for treating and / or preventing diseases with abnormal CD13 expression. The method includes: administering to a subject in need a therapeutically and / or prophylactically 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, the pharmaceutical composition or biological agent described in the seventh aspect of the present invention.

[0082] In some embodiments, the subjects include humans and non-human animals. Non-human animals include all vertebrates (such as mammals and non-mammals), such as non-human primates (such as 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 : Electrophoresis result diagram of cell broth;

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

[0085] Figure 3 : The result graph corresponding to the purity of the purified product analyzed by SEC-HPLC;

[0086] Figure 4 : The result graph of antibody titer detection. Specific implementation manners

[0087] The present invention will be further described below in conjunction with specific embodiments. The specific embodiments are only used to explain the present invention and should not be construed as a limitation to the present invention. Those of ordinary skill in the art can understand that: various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and purposes 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. Without special instructions, they can all be obtained from commercial channels. The experimental methods without specific conditions noted in the present invention are usually carried out according to the conventional conditions in the art or according to the conditions recommended by the manufacturers. In particular, the following embodiments 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 their results described in the following embodiments 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 Design, construction of expression plasmid and large-scale plasmid extraction

[0090] Construct an expression plasmid for CD13 antibody and extract the plasmid on a large scale for cell transient transfection. The specific experimental methods are as follows:

[0091] 1. Expression 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 NO: 1-3 respectively. The amino acid sequences corresponding to CDRH1-3 in the heavy chain variable region of the antibody are shown in SEQ ID NO: 4-6 respectively. See 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 antibody

[0094]

[0095] 2. Vector construction and large-scale plasmid extraction

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

[0097] Among them, the reading frame sequence of the pJD20 plasmid (signal peptide + light chain + T2A + heavy chain) is as follows:

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

[0099] Using HEK293 as the host cell for recombinant expression of antibodies, the specific experimental methods are as follows:

[0100] 1. Main materials

[0101] HEK293 cells (human embryonic kidney cells), self-preserved by our company; HEK293 serum-free medium (product number: AS-11), Tongli Haiyuan; HEK-293 expression feeding medium (product number: AS-18), Tongli Haiyuan; transfection reagent TA-293 (product number: K20001), Zhuhai Kairui; recombinant protein expression enhancer KE-293 (product number: K30001), Zhuhai Kairui; glutamine (product number: G8540-100G), Merck; expression plasmid: the plasmid (pJD20) extracted in the above steps. Cell culture flask, 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] Resuscitate HEK293 cells and perform subculture when the cell culture density reaches 3.5×10 6 / mL. According to the cell density, add fresh 293 serum-free medium to dilute the cell density to 0.4 - 0.6×10 6 / mL, and culture in a cell culture shaker (37°C, CO2 concentration 5%). Requirement: The volume of the medium does not exceed 1 / 3 of the volume of the culture flask.

[0105] (2) Transient transfection and feeding

[0106] 1) On the day of transfection, detect the 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), add it to a 50 mL centrifuge tube (tube A) pre-filled with 20 mL of 293 serum-free medium, and gently pipette to mix; take 5 mL of TA-293, add it to a 50 mL centrifuge tube (tube B) pre-filled with 20 mL of 293 serum-free medium, and gently pipette to mix;

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

[0109] 4) Slowly add the transfection reagent-plasmid complex to the cells with a pre-adjusted density using a pipette. While adding, shake the cell flask to evenly disperse the complex. After completion, transfer the flask to a shaker and start culturing under the following conditions: 5% CO2, 37 °C, 120 rpm;

[0110] 5) After culturing for 24 h (Day 1), sample to detect the cell density and viability (reserve samples), and sequentially add 50 mL of feeding medium and 6 mL of KE-293, then continue culturing;

[0111] 6) On Day 3, detect the cell density and viability (reserve samples), add 50 mL of feeding medium, and continue culturing;

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

[0113] 3. Experimental Results

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

[0115] Table 2 Monitoring Results of Cell Status during the Expression Process

[0116]

[0117]

[0118] Example 3 Affinity Chromatography

[0119] Capture the recombinant anti-CD13 antibody in the cell fermentation broth using an affinity chromatography packing material. The specific experimental method is as follows:

[0120] 1. Main Materials

[0121] Affinity chromatography packing material: Qianchun, MabPureA LX packing material (5 mL); chromatography system, InSep, Unique AutoPre100D.

[0122] 2. Experimental Method

[0123] (1) Column packing: Clean a 5 mL pre-packed column for standby. Take 5 mL of MabPureA LX packing material according to the correct glue ratio. Install the lower gasket and lower plug into the pre-packed column, pour the packing material from above to let it settle naturally. After all the packing material is installed, install 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) Loading: 1 L of the expressed product was loaded, with a loading volume of 620 mL;

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

[0128] (6) Re-equilibration: 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] The antibody with high purity was obtained through buffer exchange with G25 and ion exchange chromatography. The specific experimental method is as follows:

[0135] 1. Main materials

[0136] Bestdex G-25 packing material, Bio-Gel; Q Bestarose FF packing material, Bio-Gel; SP Bestarose FF packing material, Bio-Gel; chromatography system, EDC, Unique AutoPre 100D.

[0137] 2. Experimental method

[0138] (1) Buffer exchange with G25

[0139] 1) Column treatment: Flush with water at 5 mL / min for 10 CV;

[0140] 2) CIP: Flush with CIP solution at 8 mL / min for 10 CV;

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

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

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

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

[0145] (2) Q Purification

[0146] 1) Column packing: Clean a 5 mL pre-packed Q filler chromatography column for standby, and control the flow rate at 6 mL / min during the purification process;

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

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

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

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

[0151] (3) SP Purification

[0152] 1) Column packing: Clean a 5 mL pre-packed SP filler chromatography column for standby, and control the flow rate at 5 mL / min during the purification process;

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

[0154] 3) Sample preparation for loading: JD20-AGQ, 39 mL, dilute with water to conductivity of 2.14 mS / cm, adjust to pH 5.0, final volume 135 mL;

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

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

[0157] 6) Column treatment: Wash the column with Solution B, 6 CV.

[0158] 3. Experimental Results

[0159] SDS-PAGE electrophoresis was used to analyze each component eluted in each step (electrophoresis results are shown in Figure 2) The purity of the final cationic (SP) 10% B elution product > 90%. The concentration of the JD20-AGQS sample detected by BCA quantification was 3.28 mg / mL.

[0160] Example 5 Identification of Antibody Properties

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

[0162] 1. Main Materials

[0163] Recombinant human CD13 protein (10051-H08H), Sino Biological Inc.; Phosphate buffer, Shanghai Yuanpei; Goat Anti-Human IgG Fc / HRP (ab97225), Abcam; TMB Substrate Chromogenic Kit (CW0050S), ComWin Biotech; Multifunctional Microplate Reader (INFINITE EPLEX), Tecan; High Performance Liquid Chromatograph (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 result of the BCA method;

[0167] 2) Detection Conditions:

[0168] Chromatographic column: BioCore SEC- 7.8*300mm 3μm; Mobile phase: 900 mL of 0.1M Na2SO4 + 0.1M Na2HPO4 aqueous solution adjusted to pH 6.5 with phosphoric acid + 100 mL of acetonitrile; Detection wavelength: 210 nm; Column temperature: 30 °C; Isocratic elution; Flow rate: 0.5 mL / min; Injection volume: 40 μL; Detection time: 30 min;

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

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

[0171] (2) ELISA Detection of Antibody Titer

[0172] Conventional ELISA assay: Coat with recombinant human CD13 protein at 0.5 μg / well; block with 5% non-fat milk powder; dilute the recombinant anti-human CD13 antibody (1 g / L) to 100 mg / L and then serially dilute it 10-fold to 100 ng / L. Use antibody diluent as the negative control and incubate at 37°C for 1 h; the secondary antibody is goat anti-human IgG Fc / HRP, diluted 10,000-fold, 100 μL per well, and incubate at 37°C for 50 min; develop color with TMB, read with an ELISA reader, and analyze the results. Among them, the concentrations of the antibody are: 1: 100 mg / L; 2: 10 mg / L; 3: 1 mg / L; 4: 100 μg / L; 5: 10 μg / L; 6: 1 μg / L; 7: 100 ng / L; 8: 0 ng / L.

[0173] 3. Experimental results

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

Claims

1. An antibody against human CD13, 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 NO: 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 NO: 4-6, respectively.

2. The antibody according to claim 1, wherein The amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 7; The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:

8.

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

4. An expression vector, characterized in that The expression vector contains the nucleic acid molecule according to claim 3.

5. The expression vector according to claim 4, wherein The vector is a DNA vector and / or an RNA vector.

6. The expression vector according to claim 4, characterized in that, The vector is a plasmid, a virus-derived vector, and / or a transposon vector.

7. The expression vector according to claim 6, characterized in that, The virus-derived vector is a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a poxviral vector, and / or a herpesviral vector.

8. The expression vector according to claim 4, wherein The expression vector is the expression vector pCDNA3.4 with a reading frame of signal peptide - the light chain of the antibody according to claim 1 or 2 - T2A - the heavy chain of the antibody according to claim 1 or 2.

9. The expression vector according to claim 8, characterized in that, The signal peptide is Mouse Ig Kappa signal peptide.

10. The expression vector according to claim 8, characterized in that, The amino acid sequence of the signal peptide is shown in SEQ ID NO:

9.

11. The expression vector according to claim 8, wherein, The reading frame sequence of the expression vector is shown in SEQ ID NO:

10.

12. A recombinant host cell, characterized in that, The recombinant host cell contains the expression vector according to any one of claims 4-11.

13. The recombinant host cell according to claim 12, wherein The host cell is a mammalian cell, an insect cell, and / or a yeast cell.

14. The recombinant host cell according to claim 13, wherein The host cell is a HEK293 cell, a CHO cell, an NS0 myeloma cell, an SP2 / 0 myeloma cell, a COS cell, a BHK cell, or a Vero cell.

15. An antibody conjugate, characterized in that, The antibody conjugate is a complex formed by directly or indirectly conjugating the antibody according to claim 1 or 2 to a detectable label.

16. The antibody conjugate according to claim 15, wherein The detectable label is a bioluminescent agent or a chemiluminescent agent.

17. A cell sorting reagent, characterized in that, The cell sorting reagent is a complex formed by binding the antibody according to claim 1 or 2 to magnetic beads.

18. A detection reagent, characterized in that, The detection reagent contains the antibody according to claim 1 or 2 or the antibody conjugate according to claim 15 or 16.

19. A detection product, characterized in that, The detection product contains the antibody according to claim 1 or 2, the antibody conjugate according to claim 15 or 16, or the detection reagent according to claim 18.

20. The detection product according to claim 19, wherein, The detection product is a detection kit, a test strip, or a detection chip.

21. A method for producing the antibody according to claim 1 or 2, characterized in that, The method includes: culturing the recombinant host cell according to any one of claims 12-14, and isolating the antibody from the culture product of the recombinant host cell.

22. A method for preparing a recombinant host cell according to any one of claims 12-14, characterized in that, The method includes: introducing the expression vector according to any one of claims 4-11 into a host cell to obtain the recombinant host cell according to any one of claims 12-14.

23. A method for sorting CD13-positive cells, characterized in that, The method includes: sorting a cell population to be sorted using the cell sorting reagent according to claim 17.

24. A method for detecting CD13 in a sample to be tested for non-diagnostic and non-therapeutic purposes, characterized in that, The method includes: contacting a sample to be tested with the antibody according to claim 1 or 2, the antibody conjugate according to claim 15 or 16, the detection reagent according to claim 18, or the detection product according to claim 19 or 20, and detecting the formation of an immune complex of CD13 protein and the antibody.

25. Use of the antibody according to claim 1 or 2, the nucleic acid molecule according to claim 3, the expression vector according to any one of claims 4 - 11, or the recombinant host cell according to any one of claims 12 - 14 in the preparation of an antibody conjugate for detecting CD13.

26. Use of the antibody according to claim 1 or 2, the nucleic acid molecule according to claim 3, the expression vector according to any one of claims 4 - 11, or the recombinant host cell according to any one of claims 12 - 14 in the preparation of a cell sorting reagent for sorting CD13 - positive cells.

27. Use of the antibody according to claim 1 or 2, the nucleic acid molecule according to claim 3, the expression vector according to any one of claims 4 - 11, the recombinant host cell according to any one of claims 12 - 14, or the antibody conjugate according to claim 15 or 16 in the preparation of a detection reagent for detecting CD13.

28. Use of the antibody according to claim 1 or 2, the nucleic acid molecule according to claim 3, the expression vector according to any one of claims 4 - 11, the recombinant host cell according to any one of claims 12 - 14, the antibody conjugate according to claim 15 or 16, or the detection reagent according to claim 18 in the preparation of a detection product for detecting CD13.

29. Use of the antibody according to claim 1 or 2, the nucleic acid molecule according to claim 3, the expression vector according to any one of claims 4 - 11, the recombinant host cell according to any one of claims 12 - 14, the antibody conjugate according to claim 15 or 16, the detection reagent according to claim 18, or the detection product according to claim 19 or 20 in the detection of CD13 for non - diagnostic and non - therapeutic purposes.

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