Anti-human IgG antibody and preparation method and application thereof

Abstract

The invention discloses an anti-human IgG antibody and a preparation method and application thereof, a heavy chain variable region of the antibody comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, amino acid sequences of the heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NO: 11-13, a light chain variable region of the antibody comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3, and amino acid sequences of the light chain complementary determining regions LCDR1, LCDR2 and LCDR3 are respectively shown as SEQ ID NO: 14-16. The high-activity monoclonal antibodies combined with different subtypes of human IgG Fc are efficiently prepared by utilizing an in-vitro culture method of B cells, and in particular, the invention relates to two monoclonal antibodies which can be specifically combined with human IgG1 protein, so that a powerful tool is provided for detecting the human IgG1 protein and researching diseases related to the human IgG1 protein.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, specifically to anti-human IgG antibodies, preparation methods and uses thereof, and more particularly to antibodies that specifically bind to mammalian, especially human, IgG Fc, and preparation methods and uses thereof. Background Art

[0002] Antibodies derived from mice, rabbits, alpacas, or humans are called primary antibodies. Secondary antibodies, which bind to the primary antibody, are called anti-antibodies. Their primary function is to detect the presence of the primary antibody and amplify its signal. Secondary antibodies play an important role in experiments such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry. Traditionally, secondary antibodies are prepared by immunizing a xenogeneic animal with the antibody as the immunogen. The xenogeneic animal's immune system then produces immunoglobulins specific to the primary antibody, which are then affinity-purified to produce polyclonal antibodies. Polyclonal antibodies are mixtures of antibodies produced by different B lymphocytes that recognize multiple epitopes on an antigen molecule. After a batch is used up, further immunization is required for antibody production. Each immune response can vary depending on factors such as antigen purity and individual animal differences, leading to significant variability in purity, specificity, and potency between different batches of antibodies. In new drug development, especially for humanized antibody research, highly specific and low-background anti-IgG Fc antibodies are required. Rabbit monoclonal antibodies, due to their high affinity and low background, are a preferred 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 affinity for the research and application of related diseases. Summary of the Invention

[0003] To address the above technical problems, the present 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, the two monoclonal antibodies can specifically bind to human IgG1 protein, providing a powerful tool for detecting human IgG1 protein and studying diseases associated with human IgG1 protein.

[0004] In a first aspect, 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 complementary determining regions HCDR1, HCDR2 and HCDR3, and the amino acid sequences are shown in SEQ ID NOs: 11-13, respectively; and the light chain variable region comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3, and the amino acid sequences are shown in SEQ ID NOs: 14-16, respectively.

[0005] It is understood that the above amino acid sequence encompasses 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, which may contain mutations such as deletions, insertions and / or substitutions, wherein the sequence of the heavy chain variable region or light chain variable region differs from the reference sequence only in conservative 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 that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to 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 that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to 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; and 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 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 code.

[0010] In a preferred embodiment, the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region is 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 code.

[0011] In a preferred embodiment, it further comprises 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 kappa chain or a lambda chain constant region.

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

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

[0015] More preferably, the antibody or antigen-binding fragment thereof 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 a specific embodiment, the antibody or antigen-binding fragment thereof further comprises a human IgG1 heavy chain constant region as shown in SEQ ID NO: 21 or a variant thereof.

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

[0018] The sequence information of the above antibodies is shown in Tables 1 and 2.

[0019] Table 1 Variable region sequences

[0020]

[0021]

[0022] Table 2 Constant region sequences

[0023]

[0024] Another aspect of the present invention further provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

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

[0026] Another aspect of the present invention provides a recombinant cell, which comprises the nucleic acid molecule and / or the recombinant vector as described in the previous aspect of the present invention, or expresses the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention.

[0027] It is understood that recombinant cells refer to cells into which the vector has been introduced, 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, which comprises culturing a recombinant cell containing a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention under conditions suitable for expressing the antibody.

[0029] In a preferred embodiment, the method further comprises recovering the antibody or antigen-binding fragment thereof from the recombinant cell or culture medium.

[0030] Another aspect of the present invention further provides the use of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the nucleic acid molecule according to the above aspect of the present invention, the recombinant vector according to the above aspect of the present invention, and / or the recombinant cell according to the above aspect of the present invention in drug research and development, for example, in the preparation of products for diagnosing and treating diseases related to human IgG1-4 proteins, and in studying cell cycle and cell biology.

[0031] Another aspect of the present invention provides use of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the nucleic acid molecule according to the above aspect of the present invention, the recombinant vector according to the above aspect of the present invention, and / or the recombinant cell according to the above aspect of the present invention in the preparation of a reagent for detecting human IgG1-4 proteins for non-diagnostic purposes.

[0032] Another aspect of the present invention provides a method for detecting human IgG1-4 proteins in a sample for non-diagnostic purposes, characterized in that it comprises the following steps: contacting the sample to be tested with the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, and confirming the presence or level of human IgG1-4 proteins in the sample to be tested.

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

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

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

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

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

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

[0039] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

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

[0041] The positive progress of the present invention lies in: using human IgG Fc as the immune source, the present invention immunized New Zealand white rabbits, screened multiple specific antibody clones, and identified the binding of these antibodies to different subtypes of IgG1, IgG2, IgG3 and IgG4 Fc, and obtained human IgG Fc antibodies that can simultaneously bind to different subtypes, providing more options for commercial rabbit monoclonal antibodies and providing an efficient research and development tool for drug research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the result of immune titer test.

[0043] Figure 2 Schematic diagram of detecting the binding activity of clone supernatants at the protein level by ELISA.

[0044] Figure 3 Schematic diagram of the binding activity of antibodies 002-1 and 002-2 with human IgG1 protein.

[0045] Figure 4 Schematic diagram of the binding activity of antibodies 002-1 and 002-2 to human IgG1, IgG2, IgG3, and IgG4 Fc. DETAILED DESCRIPTION

[0046] Unless otherwise specified, the following terms shall have the meanings set forth below. Other terms or abbreviations have meanings commonly known in the art.

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

[0048] "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, an antibody fragment binds to the same antigen recognized by the intact antibody. The term "antigen-binding fragment" includes aptamers, Spiegelmers, and diabodies. The term "antigen-binding fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.

[0049] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region includes 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 the present disclosure may include: a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain; or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the present disclosure includes a polypeptide chain containing a CH3 domain. In addition, an antibody for use in the present disclosure may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As described above, those of ordinary skill in the art will appreciate that the heavy chain constant region can be modified such that it differs in amino acid sequence from a naturally occurring immunoglobulin molecule.

[0050] "Nucleic acid" or "polynucleotide" refers to a polymer molecule composed of individual nucleotides: adenine (a), cytosine (c), guanine (g), thymine (t) (or uracil (u) in RNA), such as DNA, RNA, or modifications thereof. A nucleic acid molecule can be a natural nucleic acid molecule or a synthetic nucleic acid molecule or a combination of one or more natural nucleic acid molecules and 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, ribozymes, 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 separated from at least one contaminating nucleic acid molecule. An isolated nucleic acid molecule is different from its naturally occurring form or environment. Thus, an isolated nucleic acid molecule is distinguished from a nucleic acid molecule present in its natural cell. However, an isolated nucleic acid molecule includes nucleic acid molecules contained in cells that normally express an antibody, for example, where the nucleic acid molecule is located in a chromosomal location that is different from the chromosomal location of the natural cell.

[0052] "Monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, the individual antibodies comprising the population being identical. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include multiple different antibodies directed against multiple different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0053] As used herein, a sequence "variant" refers to a sequence that differs from the indicated sequence at one or more amino acid residues but retains the biological activity of the resulting molecule.

[0054] "Amino acid" refers to an organic compound containing both an amino group and a carboxyl group, such as an α-amino acid, which can be encoded by a nucleic acid directly or in the form of a precursor. 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 natural amino acids and unnatural 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] "Conservatively substituted variants" or "conservative amino acid substitutions" are amino acid substitutions known to those skilled in the art that are made without generally altering the biological activity of the resulting molecule. In general, it is recognized by those skilled in the art that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter the biological activity. Conservative substitutions can be made with amino acids containing side chains with similar chemical properties, 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" refers to a value that is within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined (i.e., the limitations of the measurement system). Alternatively, "about" can mean a range of up to ±20%, such as ±10%, ±5%, or ±1%. Unless otherwise indicated, when a particular value appears in this application and claims, the meaning of "about" should be assumed to be within an acceptable error range for that particular value.

[0057] "Specific" binding, when referring to a ligand / receptor, antibody / antigen, or other binding pair, refers to a binding reaction that determines the presence or absence of a protein, such as TREM2, within a heterogeneous population of proteins and / or other biological agents. Thus, under specified conditions, a particular ligand / antigen binds to a particular receptor / antibody and does not bind in significant amounts to other proteins present in the sample.

[0058] The term "encoding" when applied to a polynucleotide refers to a polynucleotide that, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and / or translated to produce mRNA that produces a polypeptide and / or fragment thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0059] When used in reference to an animal, human, subject, cell, tissue, organ, or biological fluid, "administering" and "treating" refer to contacting an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administering" and "treating" can refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating a cell includes contacting an agent with a cell and contacting an agent with a fluid, wherein the fluid is contacted with the cell. "Administering" and "treating" also mean the in vitro and ex vivo treatment of a cell, for example, by an agent, diagnostic agent, binding composition, or by other cells.

[0060] The term "treating" includes amelioration or cessation of a disorder or its symptoms. Treating includes inhibiting, e.g., reducing the overall frequency of occurrence of a disorder or its symptoms.

[0061] The term "preventing" includes avoiding the initiation of a disorder or its symptoms.

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

[0063] The term "carrier" refers to a diluent, adjuvant, excipient or vehicle for treatment used in conjunction with the active ingredient. This type of pharmaceutical carrier can be a sterile liquid, such as water and oil, including oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, when the pharmaceutical composition is administered intravenously, the carrier can be water. Saline solutions and aqueous glucose solutions and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions will contain a clinically effective dose of an antibody or antibody fragment, together with a suitable carrier, to provide a dosage form suitable for the patient. The preparation should be suitable for the mode of administration. The preparation can be packaged in an ampoule, a disposable syringe or a multiple-dose vial made of glass or plastic.

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

[0065] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. It is obvious that the described embodiments are only some of the embodiments of the present invention, and are not all of the embodiments. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally measured in accordance with national standards. The experimental materials in the following examples, for which the sources are not specified, are all commercially available raw materials. The equipment used in each step of the following examples is conventional equipment. If there are no corresponding national standards, the steps are carried out in accordance with general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise specified, all parts are by weight, and all percentages are by mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention.

[0066] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of 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 they are not intended to limit the present invention.

[0067] Example 1: Animal Immunization

[0068] In order to obtain rabbit monoclonal antibodies that recognize human IgG FC antigens, the present invention selected New Zealand white rabbits for immunization, and selected commercially available human IgG1 antibodies for immunization. Each rabbit was immunized with 200 μg for the first time. During the first immunization, 1 ml of Freund's complete adjuvant and 1 ml of antigen were mixed and emulsified for half an hour. The total amount of antigen was 400 μg, and the immunization was injected at multiple points on the back. A booster immunization was performed every two weeks. The amount of antigen used for the booster immunization was 100 μg. 1 ml of Freund's incomplete adjuvant and 1 ml of antigen were mixed and emulsified, and the immunization was injected at multiple points on the back. After five immunizations, the titer of the gradient diluted immune serum was detected by the Elisa method, and the results are shown in Table 1. Figure 1 Three days before antibody screening, a single pulse immunization was performed with 50 μg of protein injected intraperitoneally. This time, the antigen did not require adjuvant emulsification, and the buffer was PBS. The spleen was harvested three days later.

[0069] The specific detection method of serum titer is as follows:

[0070] 1.1 μg / mL human IgG1 was coated on a 96-well plate, 100 μL per well, at 4°C overnight;

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

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

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

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

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

[0076] 7. Add 100 μL of anti-Rabbit [HRP] antibody diluted 1:20,000 in 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 0.1% PBST;

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

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

[0080] 11. Read the absorbance at 450 nm using a microplate reader.

[0081] Example 2: Spleen Cell Isolation

[0082] Rabbits were sacrificed by injection of gas into the ear vein. The spleens were surgically removed and placed in a sterile culture dish. The spleens were rinsed several times with PBS. Approximately one-fifth of the spleen was then removed with surgical scissors and minced. Finally, the spleen was ground into a single-cell suspension using a syringe plunger. The suspension was filtered through a 100-μm cell sieve, and the single-cell filtrate was collected. The suspension was centrifuged at 1500 rpm for 3 minutes, the supernatant discarded, and the cells resuspended in DMEM. The cell density was calculated using a cell counter.

[0083] Example 3: B cell culture and identification

[0084] To 100 μg of human IgG1 protein, add 6 μl of 20 mM activated biotin, mix gently, and incubate at room temperature for half an hour. Biotin-labeled human IgG1 protein was incubated with 1E8 spleen cells at 4°C for 2 hours. B cells were isolated using anti-biotin magnetic beads. Cells were cultured in B cell culture medium in a 96-well cell culture plate at 37°C and 5% CO2. After 14 days of culture, supernatants from clones were assayed for protein binding activity by ELISA. Clones with binding activity exceeding 3-fold background were considered positive.

[0085] The specific steps are as follows:

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

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

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

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

[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, dry it with absorbent paper, and wash three times with 260 μL 0.1% PBST;

[0092] 7. Add 100 μL of anti-Rabbit [HRP] antibody diluted 1:20,000 in 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 0.1% PBST;

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

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

[0096] 11. Read the absorbance at 450 nm using a microplate reader;

[0097] See the results Figure 2 Clones with an activity three times greater than background were further verified to determine whether they bound to the Fab or Fc end of human IgG1. Human IgG1 F(ab')2 and human Fc were used as antigens, respectively, and the ELISA method was used for verification. The results showed that two clones with higher binding activity had OD values ​​of 0.952 and 0.901, respectively. They only bound to human Fc but not F(ab')2. The two clones were named Antibody 002-1 and Antibody 002-2, respectively, and were used for subsequent experiments.

[0098] Example 4: B cell culture and identification

[0099] Positive B cell clones were harvested, and total RNA was extracted using a total RNA extraction reagent. This was reverse-transcribed into cDNA using a reverse transcription kit. The light and heavy chain variable region sequences were amplified by PCR and inserted into a T-vector. The antibody light and heavy chain sequences were obtained by sequencing. The sequencing results were analyzed using VBASE2 (http: / / www.vbase2.org / vbscAb.php). The light and heavy chain variable region sequences of Antibody 002-1 and Antibody 002-2 were obtained, as shown in 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 chain and heavy chain of the antibody were constructed into mammalian expression vectors containing the light chain constant region and the heavy chain constant region, respectively, and the correctness was verified by sequencing. The plasmid was extracted using the Tiangen Plasmid Extraction Kit. 293F cells were cultured, and 293F cells in the logarithmic growth phase were inoculated into 250mL and 250ml triangular cell culture flasks, and 50mL of culture medium was added for culture. When the cell density reached 1E6 / ml, transfection was performed, and PEI was co-transfected with the light chain expression plasmid and the heavy chain expression plasmid. The cell supernatant was collected on the 5th day of culture after transfection, centrifuged and filtered using a syringe filter. The antibody was purified by Protein A and replaced by ultrafiltration into PBS pH 7.2 buffer. The antibody concentration and purity were determined by measuring the absorbance by Nanodrop, and the purity was checked by sodium dodecyl sulfate gel electrophoresis and Coomassie staining.

[0102] Example 6: Detection of Binding Activity of Anti-human IgG1 Fc Antibody to Human IgG1 Protein

[0103] In this example, the binding activity of the anti-human IgG1 antibody obtained in Example 5 to the human IgG1 protein was verified by ELISA. The specific steps included: the coating concentration of the human IgG1 protein was 1 μg / mL, the starting concentration of antibodies 002-1 and 002-2 was 1000 nM, four-fold dilution was used, and a total of 12 concentrations were obtained. After incubation of the antigen and antibody for 3 hours, a 1:20,000 dilution of anti-rabbit-IgG-HRP antibody was added. After incubation for 1 hour, the OD450 absorbance was measured by a microplate reader. The results were as follows: Figure 3 As shown, the EC50 values ​​of the two antibodies were 4.084 and 5.508 nM, respectively.

[0104] Example 7: Detection of Antibody Binding Activity to Human IgG1, IgG2, IgG3, and IgG4 Fc

[0105] In this example, the binding activity of the anti-human IgG1 antibody obtained in Example 5 to different subtypes of IgG was verified by ELISA. The specific steps included: the coating concentration of commercially available human IgG1, IgG2, IgG3, and IgG4 Fc proteins was 1 μg / mL, and antibodies 002-1 and 002-2 were first biotinylated. Then, the antibodies were diluted in series with a starting concentration of 2000 ng / mL for a total of 10 concentrations. After incubation of the antigen and antibody for 3 hours, a 1:20,000 dilution of anti-streptavidin-HRP antibody was added. After incubation for 1 hour, the OD450 absorbance was measured by a microplate reader. The results were as follows: Figure 4 As shown, the two antibodies were analyzed to bind to human IgG1, IgG2, IgG3 and IgG4 Fc, respectively, and the binding activities were comparable.

Claims

1. An anti-human IgG antibody or 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 complementary determining regions HCDR1, HCDR2, and HCDR3, whose amino acid sequences are shown in SEQ ID NOs: 11-13, respectively, and the light chain variable region comprises light chain complementary determining regions LCDR1, LCDR2, and LCDR3, whose amino acid sequences are shown in SEQ ID NOs: 14-16, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein: The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of 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 of SEQ ID NO: 17; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of 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 of SEQ ID NO:

19.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein: 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.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, further comprising a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof; Preferably, the light chain constant region is a kappa chain or a lambda chain constant region; Preferably, the heavy chain constant region is selected from the class of IgG, IgM, IgA, IgE or IgD, More preferably, the heavy chain constant region is a heavy chain constant region selected from the IgG1, IgG2, IgG3 or IgG4 subclass; More preferably, the antibody or antigen-binding fragment thereof further comprises a human IgG1 heavy chain constant region or a variant thereof, preferably as shown in SEQ ID NO: 21, and / or a human κ light chain constant region or a variant thereof, preferably as shown in SEQ ID NO:

22.

5. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4. A recombinant vector comprising the nucleic acid molecule according to claim 5 .

7. A recombinant cell comprising the nucleic acid molecule of claim 5 and / or the recombinant vector of claim 6, or expressing the antibody or antigen-binding fragment thereof of any one of claims 1 to 4.

8. A method for producing an antibody or an antigen-binding fragment thereof, the method comprising culturing a recombinant cell comprising a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 under conditions suitable for expression of the antibody, and optionally further comprising recovering the antibody or antigen-binding fragment thereof from the recombinant cell or culture medium.

9. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the recombinant vector according to claim 6, and / or the recombinant cell according to claim 7 in drug research and development.

10. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the recombinant vector according to claim 6 and / or the recombinant cell according to claim 7 in the preparation of a reagent for detecting human IgG1-4 proteins for non-diagnostic purposes.

11. A method for detecting human IgG1-4 proteins in a sample for non-diagnostic purposes, characterized in that: The method comprises the following steps: contacting a sample to be tested with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, and confirming the presence or level of human IgG1-4 proteins in the sample to be tested.

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