Anti-human IgG antibodies, their preparation methods and uses

Abstract

This patent discloses an antibody against human IgG, its preparation method, and its uses. The heavy chain variable region of the antibody includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, with amino acid sequences shown in SEQ ID NO:11-13, respectively. The light chain variable region includes light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, with amino acid sequences shown in SEQ ID NO:14-16, respectively. This invention utilizes an in vitro B cell culture method to efficiently prepare highly active monoclonal antibodies that bind to different subtypes of human IgG Fc. Specifically, it involves two monoclonal antibodies that can specifically bind to human IgG1 protein, providing a powerful tool for detecting human IgG1 protein and studying diseases related to human IgG1 protein.

Description

Technical Field

[0001] This invention relates to the field of antibodies, specifically to antibodies against human IgG, their preparation methods and uses, and more particularly to antibodies that specifically bind to mammalian, especially human, IgG Fc, their preparation methods and applications. Background Technology

[0002] Antibodies derived primarily from mice, rabbits, alpacas, or humans are called primary antibodies. Secondary antibodies, or anti-antibodies, are antibodies that bind to the primary antibody and primarily detect its presence and amplify its signal. Secondary antibodies play a crucial role in experiments such as flow cytometry, enzyme-linked immunosorbent assays (ELISA), and immunohistochemistry. Traditional secondary antibody preparation involves using antibodies as an immunogen to immunize a xenobiotic animal. The xenobiotic animal's immune system produces immunoglobulins against the primary antibody, which are then purified through affinity testing to obtain polyclonal antibodies. Polyclonal antibodies are mixtures of antibodies produced by different B lymphocytes that recognize multiple different antigenic epitopes on the antigen molecule. After a batch is used up, repeated immunization is required for antibody preparation. Each immunization reaction varies due to factors such as antigen purity and individual animal differences, resulting in significant variations in purity, specificity, and titer between different batches of antibodies. In new drug development, especially in the research of humanized antibody drugs, high-specificity and low-background anti-IgG Fc antibodies are required. Rabbit monoclonal antibodies, due to their high affinity and low background, are a better choice. However, there is currently a lack of human IgG Fc antibodies that simultaneously bind to different subtypes, especially high-affinity recombinant rabbit monoclonal antibodies. Therefore, it is necessary to further develop rabbit monoclonal antibodies with higher activity and higher affinity for research and application in related diseases. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention utilizes an in vitro culture method for B cells to efficiently prepare highly active monoclonal antibodies that bind to different subtypes of human IgG Fc. Specifically, it involves two monoclonal antibodies that can specifically bind to human IgG1 protein, providing a powerful tool for detecting human IgG1 protein and studying diseases related to human IgG1 protein.

[0004] The first aspect of the present invention provides an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are shown in SEQ ID NO:11-13, respectively, and the light chain variable region comprises light chain complementarity-determining regions LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are shown in SEQ ID NO:14-16, respectively.

[0005] Understandably, the above-mentioned amino acid sequences encompass amino acid sequences that are at least 80% identical to themselves (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) and have the same function, and may contain mutations such as deletions, insertions, and / or substitutions, wherein the heavy chain variable region or light chain variable region sequence differs from the reference sequence only in conserved amino acid substitutions.

[0006] In a preferred embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:17, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:17, or a sequence having one or more amino acid substitutions, deletions, or insertions, or any combination thereof, compared to the amino acid sequence shown in SEQ ID NO:17.

[0007] In a preferred embodiment, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:19, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:19, or a sequence having one or more amino acid substitutions, deletions, or insertions, or any combination thereof, compared to the amino acid sequence shown in SEQ ID NO:19.

[0008] In a preferred embodiment, the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:17; the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:19.

[0009] In a preferred embodiment, the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region is either the nucleotide sequence shown in SEQ ID NO:18 or a nucleotide sequence that differs from SEQ ID NO:18 due to the degeneracy of the genetic codon.

[0010] In a preferred embodiment, the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region is either the nucleotide sequence shown in SEQ ID NO:20 or a nucleotide sequence that differs from SEQ ID NO:20 due to the degeneracy of the genetic codon.

[0011] In a preferred embodiment, it further includes a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

[0012] Preferably, the light chain constant region is a κ chain or λ chain constant region.

[0013] More preferably, the heavy chain constant region is selected from the IgG, IgM, IgA, IgE or IgD class.

[0014] More preferably, the heavy chain constant region is a heavy chain constant region selected from IgG1, IgG2, IgG3 or IgG4 subclasses.

[0015] More preferably, the antibody or its antigen-binding fragment further comprises a human IgG1 heavy chain constant region or a variant thereof and a human κ light chain constant region or a variant thereof.

[0016] In one specific embodiment, the antibody or its antigen-binding fragment further comprises a human IgG1 heavy chain constant region or a variant thereof as shown in SEQ ID NO:21.

[0017] In one specific embodiment, the antibody or its antigen-binding fragment further comprises a human κ light chain constant region or a variant thereof as shown in SEQ ID NO:22.

[0018] The antibody-related sequence information is shown in Tables 1 and 2.

[0019] Table 1 Variable Region Sequence

[0020]

[0021]

[0022] Table 2. Constant Region Sequence

[0023]

[0024] In another aspect, the present invention provides a nucleic acid molecule that encodes an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention.

[0025] Another aspect of the present invention provides a recombinant vector comprising nucleic acid molecules as described in the preceding aspect of the present invention.

[0026] Another aspect of the present invention provides recombinant cells comprising nucleic acid molecules as described in the preceding aspect of the present invention and / or recombinant vectors as described in the preceding aspect of the present invention, or expressing antibodies or antigen-binding fragments as described in the first aspect of the present invention.

[0027] It is understandable that recombinant cells refer to cells introduced into a vector, including prokaryotic cells, fungal cells, insect cells, animal cells, etc., such as Escherichia coli, yeast cells, S2 Drosophila cells, BHK cells, CHO cells, and HEK 293 cells.

[0028] Another aspect of the present invention provides a method for preparing an antibody or an antigen-binding fragment thereof, the method comprising, when suitable for antibody expression, culturing recombinant cells containing nucleic acid molecules encoding an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention.

[0029] In a preferred embodiment, the method further includes recovering the antibody or its antigen-binding fragment from the recombinant cells or culture medium.

[0030] In another aspect, the present invention also provides the application of antibodies or antigen-binding fragments thereof as described in the first aspect of the present invention, nucleic acid molecules as described in the above aspects of the present invention, recombinant vectors as described in the above aspects of the present invention, and / or recombinant cells as described in the above aspects of the present invention in drug development, for example, in the preparation of products for the diagnosis and treatment of diseases related to human IgG1-4 proteins, and in the study of cell cycle and cell biology.

[0031] In another aspect, the present invention also provides the use of antibodies or antigen-binding fragments thereof as described in the first aspect of the present invention, nucleic acid molecules as described in the above aspects of the present invention, recombinant vectors as described in the above aspects of the present invention, and / or recombinant cells as described in the above aspects of the present invention in the preparation of reagents for the detection of human IgG1-4 proteins for non-diagnostic purposes.

[0032] Another aspect of the present invention provides a method for detecting human IgG1-4 protein in a sample for non-diagnostic purposes, characterized by comprising the following steps: contacting the sample to be tested with the antibody or its antigen-binding fragment described in the first aspect of the present invention to confirm the presence or level of human IgG1-4 protein in the sample to be tested.

[0033] The aforementioned test samples cover a variety of sample types obtained from subjects and can be used for diagnosis or testing, including but not limited to blood and other biological liquid samples, solid tissue samples, including clinical samples, cells in culture media, cell supernatants, cell lysates, serum, plasma, biological fluids and tissue samples, etc.

[0034] Furthermore, the antibody or its antigen-binding fragment may be used as a coating antibody and an enzyme-labeled secondary antibody (detection antibody), etc.

[0035] The present invention also provides a product containing the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the nucleic acid molecule described in the above aspects of the present invention, the recombinant vector described in the above aspects of the present invention, and / or the recombinant cell described in the above aspects of the present invention.

[0036] In a preferred embodiment, the product includes a kit or drug conjugate containing the antibody or its antigen-binding fragment, the nucleic acid molecule, the recombinant vector, or the recombinant cell.

[0037] The above products may also include other testing reagents or reaction reagents.

[0038] In a preferred embodiment, the above product can be used in conventional detection methods for the detection of human IgG protein, including ELISA, Western blot, immunofluorescence, immunoprecipitation, etc.

[0039] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0040] Unless otherwise specified, all reagents and raw materials used in this invention are commercially available.

[0041] The positive and progressive effects of this invention are as follows: This invention uses human IgG Fc as an immune source to immunize New Zealand white rabbits, screens multiple specific antibody clones, and identifies the binding of these antibodies to different subtypes of IgG1, IgG2, IgG3 and IgG4 Fc. This results in the acquisition of human IgG Fc antibodies that can bind to different subtypes simultaneously, providing more options for commercial rabbit monoclonal antibodies and providing an efficient research tool for drug development. Attached Figure Description

[0042] Figure 1 This is a graph showing the results of the immunogenicity test.

[0043] Figure 2 This is a schematic diagram illustrating the detection of binding activity of clonal supernatant at the protein level using ELISA.

[0044] Figure 3 This is a schematic diagram illustrating the binding activity of antibodies 002-1 and 002-2 with human IgG1 protein.

[0045] Figure 4 This diagram illustrates the binding activity of antibodies 002-1 and 002-2 with human IgG1, IgG2, IgG3, and IgG4 Fc. Detailed Implementation

[0046] Unless otherwise stated, the following terms shall have the meanings described below. Other terms or abbreviations shall have meanings known in the art.

[0047] "Antibody" refers to any form of antibody that exhibits a desired biological activity (e.g., inhibiting the binding of a ligand to its receptor or by inhibiting receptor signal transduction induced by a ligand). Therefore, "antibody" is used in its broadest sense and is specifically defined to include, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and multispecific antibodies (e.g., bispecific antibodies), fully human, humanized, primate-derived, chimeric antibodies, single-chain antibodies, etc.

[0048] An "antigen-binding fragment" refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of its structure, the antibody fragment binds to the same antigen recognized by the intact antibody. The term "antigen-binding fragment" includes aptamers, mirror isoforms, and bivalent antibodies. The term "antigen-binding fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by forming a complex with a specific antigen.

[0049] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from the immunoglobulin heavy chain. A polypeptide containing a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (e.g., an upper hinge region, a middle hinge region, and / or a lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in this disclosure may comprise: a polypeptide chain containing a CH1 domain; a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, and a CH2 domain; a polypeptide chain containing both a CH1 domain and a CH3 domain; a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, and a CH3 domain; or a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of this disclosure comprises a polypeptide chain containing a CH3 domain. Furthermore, antibodies for use in this disclosure may lack at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). As described above, those skilled in the art will understand that the heavy chain constant region can be modified to differ from naturally occurring immunoglobulin molecules in its amino acid sequence.

[0050] "Nucleic acid" or "polynucleotide" refers to a polymer molecule composed of a single nucleotide: adenine (a), cytosine (c), guanine (g), thymine (t) (or uracil (u) in RNA), such as DNA, RNA, or modifications thereof. Nucleic acid molecules can be natural or synthetic nucleic acid molecules, or a combination of one or more natural nucleic acid molecules with one or more synthetic nucleic acid molecules. Examples of nucleic acids include, but are not limited to: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribonuclease, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.

[0051] A “nucleic acid molecule” is a nucleic acid molecule that has been identified and isolated from at least one contaminating nucleic acid molecule. The isolated nucleic acid molecule differs from its naturally occurring form or environment. Therefore, the isolated nucleic acid molecule is distinct from the nucleic acid molecule present in its natural cells. However, the isolated nucleic acid molecule includes nucleic acid molecules contained in cells that normally express antibodies, for example, where the chromosomal location of the nucleic acid molecule differs from its chromosomal location in the natural cell.

[0052] "Monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, the individual antibodies constituting said group being identical. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, unlike conventional (polyclonal) antibody preparations, which typically include multiple different antibodies targeting multiple different determinants (epitopes), each monoclonal antibody targets only a single determinant on the antigen.

[0053] The sequence “variant” used in this article refers to a sequence that differs from the sequence shown at one or more amino acid residues but retains the biological activity of the resulting molecule.

[0054] Amino acids are organic compounds that contain both amino and carboxyl groups, such as α-amino acids, which can be encoded by nucleic acids directly or in their precursor form. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). The fact that the same amino acid can be encoded by different codons is called "degeneracy of the genetic code." Amino acids include both natural and non-natural amino acids. Natural amino acids include alanine (three-letter code: ala, one-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0055] "Variations of conserved substitutions" or "conserved amino acid substitutions" refer to amino acid substitutions known to those skilled in the art that such substitutions generally do not alter the biological activity of the resulting molecule. Generally, it is generally accepted by those skilled in the art that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its biological activity. Conserved substitutions can be made by amino acids containing chemically similar side chains, such as: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid.

[0056] As used herein, the term "about" means a numerical value within an acceptable margin of error for a specific value determined by a person skilled in the art, the numerical value depending in part on how it is measured or determined (i.e., the limits of the measurement system). Alternatively, "about" may mean a range of up to ±20%, such as ±10%, ±5%, or ±1%. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of "about" should be assumed to be within an acceptable margin of error for that specific value.

[0057] When referring to ligand / receptor, antibody / antigen, or other binding pairs, "specific" binding means determining the presence of a binding reaction of the protein, such as TREM2, within a heterogeneous population of proteins and / or other biological reagents. Therefore, under specified conditions, a particular ligand / antigen binds to a specific receptor / antibody and does not bind in significant amounts to other proteins present in the sample.

[0058] When applied to polynucleotides, the term "encoding" refers to a polynucleotide that, if in its natural state or when manipulated by methods known to those skilled in the art, can be transcribed and / or translated to produce mRNA containing a polypeptide and / or fragments thereof, is called "encoding" a polypeptide. The antisense strand is the complement of this nucleic acid, and the coding sequence can be deduced from it.

[0059] When "giving" and "treating" are used to refer to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, it means contacting an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Giving" and "treating" can refer to, for example, methods of treatment, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating cells includes contacting a reagent with cells and contacting a reagent with a fluid, wherein the fluid contacts the cells. "Giving" and "treating" also mean, for example, in vitro and ex vivo treatment of cells by means of a reagent, diagnostic agent, conjugated composition, or other cells.

[0060] The term "treatment" includes the improvement or cessation of a condition or its symptoms. Treatment includes suppression, such as reducing the overall frequency of attacks of a condition or its symptoms.

[0061] The term "prevention" includes avoiding the initial stages of a disease or its symptoms.

[0062] In some implementations, the term "pharmaceuticalally acceptable carrier" refers to a substance approved by a government regulatory agency or listed in another recognized pharmacopoeia for use in animals (particularly for humans). Furthermore, "pharmaceuticalally acceptable carrier" will generally be any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation adjuvant.

[0063] The term "carrier" refers to a diluent, adjuvant, excipient, or carrier used in conjunction with an active ingredient for therapeutic purposes. Such drug carriers can be sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, the carrier can be water when the drug composition is administered intravenously. Saline solutions, glucose solutions, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Examples of suitable drug carriers are described in EWMartin's Remington's Pharmaceutical Sciences, which are incorporated herein by reference. Such compositions will contain a clinically effective dose of an antibody or antibody fragment, along with a suitable carrier, to provide a dosage form suitable for the patient. The formulation should be suitable for the mode of administration. The formulation can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0064] The antibodies or antigen-binding fragments thereof of the present invention include their salt forms. Pharmaceutically acceptable salts include those derived from anions such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those derived from cations such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0065] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental materials in the following embodiments that do not specify their source are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0066] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0067] Example 1: Animal Immunization

[0068] To obtain rabbit monoclonal antibodies recognizing the human IgG FC antigen, this invention selected New Zealand White rabbits for immunization using commercially available, conventional human IgG1 antibodies. For the initial immunization, each rabbit received 200 μg of antigen, emulsified with 1 ml of Freund's complete adjuvant and 1 ml of antigen over half an hour, resulting in a total antigen dose of 400 μg. The immunization was administered via multiple injections at the back. Booster immunizations were given every two weeks, with a dose of 100 μg of antigen, emulsified with 1 ml of Freund's incomplete adjuvant and 1 ml of antigen, and administered via multiple injections at the back. After five immunizations, the titers of the serially diluted immune serum were measured using an ELISA method. The results are shown below. Figure 1 Three days before antibody screening, a single pulse immunization was administered via intraperitoneal injection of 50 μg of protein. No adjuvant emulsification was required for this antigen, and PBS was used as the buffer. Spleens were harvested three days later.

[0069] The specific methods for detecting serum titer are as follows:

[0070] 1.1 μg / mL human IgG1 was coated onto 96-well plates, 100 μL per well, and incubated overnight at 4°C.

[0071] 2. Wash each well once with 260 μL of 0.1% PBST;

[0072] 3. Discard the supernatant, add 100 μL of DPBS-3% BSA to each well, and incubate at room temperature for 1 hour;

[0073] 4. Wash twice with 260 μL of 0.1% PBST per well;

[0074] 5. Dilute the serum at an initial ratio of 1:1000, then serially dilute to a total of 12 concentrations. Add 100 μL of the diluted supernatant to each well and incubate at room temperature for 3 hours.

[0075] 6. Discard the supernatant, blot dry with absorbent paper, and wash three times with 260 μL of 0.1% PBST;

[0076] 7. Add 100 μL of anti-Rabbit [HRP] antibody diluted 1:20000 with DPBS to each well and incubate at room temperature for 1.5 hours;

[0077] 8. Discard the supernatant and wash three times with 260 μL of 0.1% PBST;

[0078] 9. Add 100 μL TMB to each well, and shake to develop color for 5 min in the dark at room temperature;

[0079] 10. Add 50 μL of 2M HCl stop solution to each well;

[0080] 11. Use an ELISA reader to read the absorbance at 450 nm.

[0081] Example 2: Spleen cell isolation

[0082] Rabbits were euthanized via gas injection into the marginal ear vein. The spleen was surgically removed and placed in a sterile cell culture dish, then rinsed several times with PBS. Approximately one-fifth of the spleen was then surgically removed and minced. Finally, the spleen was ground into a single-cell suspension using a syringe core. The suspension was then filtered through a 100 μm cell strainer, and the single-cell filtrate was collected. The cells were centrifuged at 1500 rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended in DMEM. Cell density was determined using a cell counting chamber.

[0083] Example 3: B cell culture and identification

[0084] 100 μg of human IgG1 protein was added to 6 μl of 20 mM activated biotin, and the mixture was gently mixed and reacted at room temperature for half an hour. Biotin-labeled human IgG1 protein was then co-incubated with 1E8 spleen cells at 4°C for 2 hours, followed by B cell sorting using anti-Biotin magnetic beads. B cells were cultured in 96-well cell culture plates at 37°C and 5% CO2 using B cell culture medium. After 14 days of culture, the binding activity of the clonal supernatant was detected at the protein level using ELISA; clones with a protein binding activity greater than 3 times the background level were considered positive.

[0085] The specific steps are as follows:

[0086] 1.1 μg / mL human IgG1 was coated onto a 96-well plate, 100 μL per well, and incubated overnight at 4°C.

[0087] 2. Wash each well once with 260 μL of 0.1% PBST;

[0088] 3. Discard the supernatant, add 100 μL of DPBS-3% BSA to each well, and incubate at room temperature for 1 hour;

[0089] 4. Wash twice with 260 μL of 0.1% PBST per well;

[0090] 5. Add 100-100 μL of B cell culture supernatant to each well and incubate at room temperature for 2 hours;

[0091] 6. Discard the supernatant, blot dry with absorbent paper, and wash three times with 260 μL of 0.1% PBST;

[0092] 7. Add 100 μL of anti-Rabbit [HRP] antibody diluted 1:20000 with DPBS to each well and incubate at room temperature for 1.5 hours;

[0093] 8. Discard the supernatant and wash three times with 260 μL of 0.1% PBST;

[0094] 9. Add 100 μL TMB to each well, and shake to develop color for 5 min in the dark at room temperature;

[0095] 10. Add 50 μL of 2M HCl stop solution to each well;

[0096] 11. Use an ELISA reader to read the absorbance at 450 nm;

[0097] See results Figure 2 To further verify whether clones with binding activity greater than 3 times the background level bound to the Fab terminus or Fc of human IgG1, human IgG1 F(ab')2 and human Fc were used as antigens and verified using ELISA. The results showed that the two clones with high binding activity had OD values ​​of 0.952 and 0.901, respectively, and bound only human Fc, not F(ab')2. The two clones were named Antibody 002-1 and Antibody 002-2, respectively, for subsequent experiments.

[0098] Example 4: B cell culture and identification

[0099] B-cell positive clones were collected, and total RNA was extracted using a total RNA extraction reagent. The RNA was then reverse transcribed into cDNA using a reverse transcription kit. The light and heavy chain variable regions were amplified by PCR and constructed into a T-vector. Sequencing was then performed to obtain the antibody light and heavy chain sequences. The sequencing results were analyzed using VBASE2 (http: / / www.vbase2.org / vbscAb.php), yielding the light and heavy chain variable region sequences of antibodies 002-1 and 002-2 (see Table 1 above). The constant region sequences are shown in Table 2 above.

[0100] Example 5: Expression and purification of monoclonal antibodies

[0101] The light and heavy chains of the antibody were constructed into mammalian expression vectors containing the constant regions of the light and heavy chains, respectively, and their correctness was verified by sequencing. Plasmids were extracted using the Tiangen Plasmid Large-Scale Extraction Kit. 293F cells were cultured, and after reaching the logarithmic growth phase, they were seeded into 250mL and 250mL triangular cell culture flasks, and 50mL of culture medium was added. When the cell density reached 1E6 / ml, transfection was performed, with PEI co-transfecting both the light and heavy chain expression plasmids. Cell supernatant was collected on day 5 post-transfection culture, centrifuged, and filtered using a syringe filter. The antibody was purified with Protein A and then replaced with PBS pH 7.2 buffer via ultrafiltration. Antibody concentration and purity were determined by Nanodrop absorbance measurement, and purity was checked by sodium dodecyl sulfate gel electrophoresis and Coomassie staining.

[0102] Example 6: Detection of the binding activity of anti-human IgG1 Fc antibody to human IgG1 protein

[0103] This embodiment verifies the binding activity of the anti-human IgG1 antibody obtained in Example 5 to human IgG1 protein using an ELISA method. The specific steps include: the coating concentration of human IgG1 protein is 1 μg / mL; the initial concentrations of antibodies 002-1 and 002-2 are 1000 nM, diluted four times to obtain 12 concentrations; after incubating the antigen and antibody for 3 hours, anti-rabbit-IgG-HRP antibody diluted 1:20000 is added; after incubation for 1 hour, the OD450 absorbance is detected using a microplate reader. The results are as follows: Figure 3 As shown, the EC50 values ​​of the two antibodies were 4.084 and 5.508 nM, respectively, according to the analysis.

[0104] Example 7: Detection of antibody binding activity with human IgG1, IgG2, IgG3 and IgG4 Fc

[0105] This embodiment verifies the binding activity of the anti-human IgG1 antibody obtained in Example 5 to different IgG subtypes using an ELISA method. The specific steps include: coating with commercially available human IgG1, IgG2, IgG3, and IgG4 Fc proteins at a concentration of 1 μg / mL; biotinylation of antibodies 002-1 and 002-2; serial dilution at an initial concentration of 2000 ng / mL, resulting in 10 concentrations; incubation of antigen and antibody for 3 hours; addition of a 1:20000 dilution of anti-streptomycin-HRP antibody; incubation for 1 hour; and detection of the OD450 absorbance using an ELISA reader. The results are as follows: Figure 4 As shown, analysis revealed that the two antibodies bind to human IgG1, IgG2, IgG3, and IgG4 Fc, respectively, and their binding activities are comparable.

Claims

1. An antibody against human IgG or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are shown in SEQ ID NO: 11-13, and the light chain variable region comprises light chain complementarity-determining regions LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are shown in SEQ ID NO: 14-16.

2. The antibody or its antigen-binding fragment according to claim 1, wherein: The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17 or a sequence having one or more amino acid substitutions, deletions, or insertions, or any combination thereof, compared to the amino acid sequence shown in SEQ ID NO: 17; and / or, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 19 or a sequence having one or more amino acid substitutions, deletions, or insertions, or any combination thereof, compared to the amino acid sequence shown in SEQ ID NO:

19.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 17, and / or the light chain variable region comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:

19.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein: The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO: 17; the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:

19.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, characterized in that, It further includes heavy chain constant regions, light chain constant regions, or combinations thereof.

6. The antibody or its antigen-binding fragment according to claim 5, characterized in that, The light chain constant region is the constant region of the κ chain or the λ chain.

7. The antibody or its antigen-binding fragment according to claim 5, characterized in that, The heavy chain constant region is selected from the IgG, IgM, IgA, IgE or IgD class.

8. The antibody or its antigen-binding fragment according to claim 7, characterized in that, The heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3 or IgG4 subclasses.

9. The antibody or its antigen-binding fragment according to claim 5, characterized in that, The antibody or its antigen-binding fragment further comprises a human IgG1 heavy chain constant region or a variant thereof, and / or a human κ light chain constant region or a variant thereof.

10. The antibody or its antigen-binding fragment according to claim 5, characterized in that, The heavy chain constant region is shown as SEQ ID NO: 21, and / or the light chain constant region is shown as SEQ ID NO:

22.

11. A nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1-10.

12. A recombinant vector comprising the nucleic acid molecule of claim 11.

13. A recombinant cell comprising the nucleic acid molecule of claim 11 and / or the recombinant vector of claim 12, or expressing the antibody or antigen-binding fragment of any one of claims 1-10.

14. A method for preparing an antibody or an antigen-binding fragment thereof, the method comprising, when suitable for antibody expression, culturing recombinant cells comprising a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof of any one of claims 1-10, optionally further comprising recovering the antibody or antigen-binding fragment thereof from the recombinant cells or culture medium.

15. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-10, the nucleic acid molecule according to claim 11, the recombinant vector according to claim 12, and / or the recombinant cell according to claim 13 in the preparation of a reagent for detecting human IgG1-4 protein for non-diagnostic purposes.

16. A method for detecting human IgG1-4 protein in a sample for non-diagnostic purposes, characterized in that, The method includes the following steps: contacting the sample to be tested with the antibody or its antigen-binding fragment as described in any one of claims 1-10 to confirm the presence or level of human IgG1-4 protein in the sample to be tested.

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