High affinity rabbit monoclonal antibodies against dupilumab and uses thereof

Abstract

The application discloses a high-affinity rabbit monoclonal antibody against dupilumab and an application thereof, and relates to the technical field of monoclonal antibody preparation.The high-affinity rabbit monoclonal antibody provided by the application is a first antibody or a second antibody;the amino acid sequence of the heavy chain of the first antibody is shown in SEQ ID NO.8, and the amino acid sequence of the light chain is shown in SEQ ID NO.9;the amino acid sequence of the heavy chain of the second antibody is shown in SEQ ID NO.17, and the amino acid sequence of the light chain is shown in SEQ ID NO.18.The application also provides nucleic acid molecules for encoding the rabbit monoclonal antibody, a recombinant strain and other biological materials, and a drug and a product for detecting dupilumab.The rabbit monoclonal antibody provided by the application has high affinity, detection sensitivity and specificity for dupilumab, and can be used for diagnosing the total drug concentration and free drug concentration of dupilumab (DUPILUMAB) in the body of an injected patient; and has no cross reaction with human IgG4 and human total IgG. Dupilumab ) in the body of an injected patient; and has no cross reaction with human IgG4 and human total IgG.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibody preparation, and in particular to a high-affinity rabbit monoclonal antibody resistant to dupilumab and applications thereof. Background Art

[0002] The interleukin-4 receptor (IL-4R, also known as CD124; IL4Rα) is a type I transmembrane protein and a member of the interleukin family. Human IL4Rα is 825 amino acids long and comprises a signal peptide, an extracellular domain, a transmembrane domain, and an intracellular domain. IL4Rα regulates IgE production, chemokine production, and mucus production by binding to IL-4 and IL-13. IL4Rα plays a key role in the development of allergic reactions and asthma.

[0003] Dupilumab ( Dupilumab ) is a fully human IL-4Rα monoclonal antibody jointly developed by Sanofi and Regeneron. It can specifically bind to the IL-4Rα subunit, competitively bind to the IL-4 and IL-13 / IL-13Rα1 complex, block IL-4 and IL-13-induced IL-4Rα-mediated signal transduction, block the type 2 inflammatory pathway, reduce the pathological response of type 2 inflammation, and thus inhibit the type 2 inflammatory response.

[0004] Based on the above research results, Dupilumab ( Dupilumab ) is a fully humanized drug for the treatment of moderate to severe atopic dermatitis, moderate to severe asthma and chronic rhinitis in children and adults. During clinical treatment, monitoring the blood concentration of antibody drugs is very important for the efficacy and safety data of the drugs. Therefore, a method for highly sensitive detection of dupilumab ( Dupilumab ) blood drug concentration detection technology is of great significance. Summary of the Invention

[0005] The present invention provides a high-affinity rabbit monoclonal antibody against Dupilumab and its application. The antibody is prepared using Dupilumab as an immunogen and a monoclonal antibody development technology based on single B lymphocyte screening and culture. The monoclonal antibody has high affinity for Dupilumab. Dupilumab ) has high detection sensitivity. This is achieved through the following technologies.

[0006] A high-affinity rabbit monoclonal antibody against dupilumab, wherein the high-affinity rabbit monoclonal antibody is a first antibody or a second antibody;

[0007] The amino acid sequence of the complementarity determining region CDR1 on the heavy chain of the first antibody is shown in SEQ ID NO.1;

[0008] the amino acid sequence of the complementarity determining region CDR2 on the heavy chain of the first antibody is as shown in SEQ ID NO. 2;

[0009] the amino acid sequence of the complementarity determining region CDR3 on the heavy chain of the first antibody is as shown in SEQ ID NO. 3;

[0010] the amino acid sequence of the complementarity determining region CDR1 on the light chain of the first antibody is as shown in SEQ ID NO. 4;

[0011] the amino acid sequence of the complementarity determining region CDR2 on the light chain of the first antibody is GAS;

[0012] the amino acid sequence of the complementarity determining region CDR3 on the light chain of the first antibody is as shown in SEQ ID NO. 5;

[0013] the amino acid sequence of the complementarity determining region CDR1 on the heavy chain of the second antibody is as shown in SEQ ID NO. 10;

[0014] the amino acid sequence of the complementarity determining region CDR2 on the heavy chain of the second antibody is as shown in SEQ ID NO. 11;

[0015] the amino acid sequence of the complementarity determining region CDR3 on the heavy chain of the second antibody is as shown in SEQ ID NO. 12;

[0016] the amino acid sequence of the complementarity determining region CDR1 on the light chain of the second antibody is as shown in SEQ ID NO. 13;

[0017] the amino acid sequence of the complementarity determining region CDR2 on the light chain of the second antibody is AAS;

[0018] the amino acid sequence of the complementarity determining region CDR3 on the light chain of the second antibody is as shown in SEQ ID NO. 14.

[0019] The present application uses an immunogen to prepare the above-mentioned anti-Dupilumab (Dupixent®) Dupilumab rabbit monoclonal antibody, which is a full human monoclonal antibody (IgG4 type) targeting interleukin-4 receptor subunit alpha (IL-4R-alpha) expressed for preparation; the preparation method is a monoclonal antibody development technology based on single B lymphocyte screening and culture.

[0020] Further, the amino acid sequence of the heavy chain variable region of the first antibody is as shown in SEQ ID NO. 6; the amino acid sequence of the light chain variable region of the first antibody is as shown in SEQ ID NO. 7.

[0021] Further, the amino acid sequence of the heavy chain of the first antibody is shown as SEQ ID NO. 8; the amino acid sequence of the light chain of the first antibody is shown as SEQ ID NO. 9.

[0022] Further, the amino acid sequence of the heavy chain variable region of the second antibody is shown as SEQ ID NO. 15; the amino acid sequence of the light chain variable region of the second antibody is shown as SEQ ID NO. 16.

[0023] Further, the amino acid sequence of the heavy chain of the second antibody is shown as SEQ ID NO. 17; the amino acid sequence of the light chain of the second antibody is shown as SEQ ID NO. 18.

[0024] The present application also provides a substance comprising any one of the following aspects:

[0025] (1) a nucleic acid molecule or a vector comprising the nucleic acid molecule, wherein the nucleic acid encodes the high-affinity rabbit monoclonal antibody described above;

[0026] (2) an engineered cell or a recombinant strain, wherein the engineered cell or the recombinant strain is used for expressing the high-affinity rabbit monoclonal antibody described above;

[0027] (3) an antibody derivative, wherein the antibody derivative comprises the high-affinity rabbit monoclonal antibody described above;

[0028] (4) a pharmaceutical composition, wherein the pharmaceutical composition comprises a therapeutically effective amount of the high-affinity rabbit monoclonal antibody described above, or the antibody derivative;

[0029] (5) a detection product, wherein the detection product comprises the high-affinity rabbit monoclonal antibody described above, or the nucleic acid molecule, or the vector, or the derivative of the high-affinity rabbit monoclonal antibody.

[0030] Further, the detection product is a competitive ELISA detection kit for detecting dupilumab.

[0031] Further, in the competitive ELISA detection kit for detecting dupilumab, the first antibody is a competitive antibody, and the second antibody is a non-competitive antibody.

[0032] The present application also provides the use of the high-affinity rabbit monoclonal antibody described above for detecting dupilumab, which is not for the purpose of disease diagnosis and treatment; or, for preparing a product for detecting dupilumab; or, for preparing a product for regulating the activity of dupilumab.

[0033] Further, the product is a detection reagent, a detection kit, a detection test strip, a detection probe, or a detection chip.

[0034] The present invention provides a method for detecting dupilumab ( Dupilumab ) products and methods, all of which use the anti-duplicitumab (first antibody and / or second antibody) provided by the present invention. The claim of "not for the purpose of disease diagnosis and treatment" in the present invention means that except for clinical use for diagnosis or detection of dupilumab ( Dupilumab ) other applications and methods besides therapeutic effects, such as scientific research in the laboratory. Of course, the high affinity rabbit monoclonal antibody provided by the present invention can detect dupilumab ( Dupilumab ) in clinical practice, it can also be used for qualitative and quantitative detection of dupilumab ( Dupilumab ) scene.

[0035] Compared with the prior art, the present invention is beneficial in that:

[0036] 1. The present invention provides anti-duplimab ( Dupilumab ) high affinity rabbit monoclonal antibody, the affinity of the primary antibody and the secondary antibody was determined using a Biacore instrument, the primary antibody and Dupilumab ( Dupilumab ) The affinity constant of the binding is 7.36×10 -11 M, secondary antibody and dupilumab ( Dupilumab ) The affinity constant of the binding is 9.45×10 -11 M; Dupilumab ( Dupilumab ) or preparation of a test for dupilumab ( Dupilumab ) products.

[0037] 2. The present invention also develops a highly sensitive and specific anti-duplimab ( Dupilumab ) competitive enzyme-linked immunosorbent assay method has important significance in clinical diagnosis and scientific research applications. The present invention provides Dupilumab ( Dupilumab ) Competitive enzyme-linked immunosorbent assay method, the competitive antibody is the first antibody in the present invention, and the non-competitive antibody is the second antibody. When the standard sample is Dupilumab ( Dupilumab ), the IC50 of the first antibody was 64.59 ng / mL, and the second antibody showed no competition. The established method can be used to diagnose dupilumab ( Figure 1 ) Total drug concentration and free drug concentration in the injected patients; no cross-reaction with human IgG4 and total human IgG. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 2 This is a diagram of serum titers after animal immunization in Example 1.

[0039] Figure 3 The results of sorting of B lymphocytes in Example 1.

[0040] Figure 4 The results of affinity detection of different concentrations of the first antibody in Example 2.

[0041] Figure 5 The results of affinity detection of different concentrations of the first antibody in Example 2.

[0042] Figure 6 The results of affinity detection of different concentrations of the first antibody in Example 2.

[0043] Figure 7 The standard curve (inhibition rate curve) of the competitive enzyme-linked immunoassay method using the first antibody and the second antibody in Example 3.

[0044] Figure 8 The results of specificity detection of the indirect enzyme-linked immunoassay method using the first antibody and the second antibody in Example 4.

[0045] Figure 9 The results of stability test of the first antibody in Example 4.

[0046] Dupilumab The results of stability test of the second antibody in Example 4. DETAILED DESCRIPTION

[0047] The technical solutions of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0048] In the embodiments of the present application, the term "nucleic acid (molecule)" can be a nucleic acid including coding, or a nucleic acid further including additional coding and / or non-coding sequences. The DNA form of the nucleic acid of the present application includes but is not limited to cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The nucleic acid coding the high-affinity rabbit monoclonal antibody or the antigen-binding fragment thereof described in the present application includes but is not limited to a coding sequence coding only the structure of the mature high-affinity rabbit monoclonal antibody or the antigen-binding fragment thereof; a coding sequence coding the mature high-affinity rabbit monoclonal antibody or the antigen-binding fragment thereof and various additional coding sequences; a coding sequence (and optional additional coding sequences) coding the structure of the mature high-affinity rabbit monoclonal antibody or the antigen-binding fragment thereof and non-coding sequences, etc.

[0049] In some embodiments of the present invention, the full-length nucleic acid sequence of the high-affinity rabbit monoclonal antibody or fragments thereof can generally be obtained by PCR amplification, recombination, or synthetic methods. Once the relevant sequence is obtained, recombinant methods can be used to obtain the relevant sequence in large quantities. Generally, the relevant sequence is cloned into a vector, then transferred into cells, and then isolated from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, high-affinity rabbit monoclonal antibodies, and antigen-binding fragments thereof, etc.) referred to in the present invention include biomolecules in isolated form.

[0050] The vectors include viral vectors (such as adenoviral vectors, retroviral vectors, adeno-associated viral vectors) and non-viral vectors (plasmids, transposon vectors).

[0051] In some preferred embodiments, the vector is an expression vector.

[0052] The terms "vector," "cloning vector," "recombinant vector," or "expression vector" refer to a vector that can introduce DNA or RNA sequences into a host cell in a manner that transforms the host and promotes the expression (e.g., transcription and translation) of the introduced sequence.

[0053] Other examples of plasmids include replicating plasmids comprising an origin of replication, or integrating plasmids.

[0054] The vector can be a recombinant expression vector or a cloning vector. The vector provided by the present invention (e.g., an expression vector) comprises a nucleic acid sequence provided by the present invention encoding an antibody, at least one promoter operably linked to the nucleic acid sequence, and / or at least one selective marker.

[0055] Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses, lambda phage, M13 phage, plasmids, and the like.

[0056] The "modified cell" provided by the present invention refers to the introduction of a "foreign" (i.e., external or extracellular) gene or DNA or RNA sequence into a host cell so that the host cell expresses the introduced gene or sequence to produce a substance of interest, usually a protein encoded by the gene or introduced sequence. The host cell that receives and expresses the introduced DNA or RNA has been "modified".

[0057] In a specific embodiment of the present invention, the host cells include but are not limited to mammalian cells, insect cells, plant cells, fungal cells, prokaryotic cells, etc.

[0058] The antibody derivative provided by the present application comprises the high-affinity rabbit monoclonal antibody provided by the present application, and is directly or indirectly connected to a connectable object to form a complex.

[0059] The connectable object comprises a detectable label, a drug, a toxin, a cytokine, a radionuclide or an enzyme.

[0060] In a specific embodiment of the present application, the connectable object is selected from a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product, a radionuclide, a biological toxin, a cytokine (such as IL-2, etc.), an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle / nanorod, a viral particle, a liposome, a nanomagnetic particle, a prodrug activating enzyme (such as DT-diaphorase (DTD) or benzophenone hydrolase-like protein (BPHL), a chemotherapeutic agent (such as cisplatin), or any form of nanoparticle, etc.

[0061] The term "detectable label" refers to any moiety that generates a measurable signal by a change in the optical, electrical or other physical indicia of the state of the molecule to which the moiety is coupled. Such physical indicia include optical, photochemical, biochemical, immunochemical, electromagnetic, radiochemical and chemical means, such as, but not limited to, fluorescence, chemiluminescence and electrochemiluminescence, etc. A "direct label" when used in reference to a labeled detection agent is a detectable label attached to the detection agent by any means. An "indirect label" when used in reference to a labeled detection agent is a detectable label that specifically binds to the detection agent. Thus, an indirect label includes moieties that are specific binding partners of the detection agent. Biotin and avidin are examples of such moieties employed, for example, by contacting a biotinylated antibody with labeled avidin to produce an indirectly labeled antibody.

[0062] The drug provided by the present application comprises a pharmaceutically acceptable carrier or excipient. In some embodiments, the excipient comprises a pharmaceutically acceptable solvent, dispersant, additive, plasticizer, etc. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable carrier medium. The prepared pharmaceutical composition can be administered by a conventional route, including but not limited to intratumoral, intraperitoneal, intravenous or local administration.

[0063] In other embodiments, the pharmaceutical composition is an aqueous pharmaceutical composition (e.g., an aqueous solution), or the high-affinity rabbit monoclonal antibody is provided with a salt in water without a buffer, or contains an aqueous buffer or other types of solvents (e.g., organic solvents).

[0064] The term "effective amount" as used herein refers to an amount sufficient to achieve a beneficial or desired result. A "therapeutically effective amount" is an amount that achieves the desired therapeutic effect. This amount may be the same as or different from a prophylactically effective amount, which is the amount required to prevent the onset of a disease or disease symptoms. An effective amount may be administered, applied, or dosed once or more. The "therapeutically effective amount" (i.e., effective dose) of a therapeutic compound depends on the therapeutic compound selected. For example, the composition may be administered from one or more times daily to one or more times weekly, to one or more times monthly, to one or more times annually. Certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or condition, previous treatments, the subject's general health and / or age, and the presence of other diseases.

[0065] Example 1: Anti-duplimab ( Figure 1 ) Preparation of rabbit monoclonal antibodies

[0066] 1. Animal immunization

[0067] In order to obtain rabbit monoclonal antibodies that recognize dupilumab, the present invention used dupilumab as an immunogen to immunize four New Zealand white rabbits (provided by the Hubei Provincial Experimental Animal Research Center), labeled 1#, 2#, 3#, and 4#, respectively; each rabbit was immunized with 250 μg;

[0068] For the first immunization, the immunogen was mixed with an equal amount of complete Freund's adjuvant to form an emulsion, which was injected subcutaneously at multiple points on the abdomen and back. Two weeks apart, 125 μg of the immunogen was mixed with an equal amount of incomplete Freund's adjuvant to form an emulsion, which was injected subcutaneously at multiple points on the abdomen and back for two booster immunizations.

[0069] After three immunizations, the serum titer was determined by ELISA. Rabbits with high serum titer were given a booster immunization with 125 μg of immunogen injected subcutaneously at multiple points. The spleen was harvested three days later.

[0070] like Figure 2 As shown, in each dilution multiple, the serum titer was the highest in 2# New Zealand white rabbit, so 2# New Zealand white rabbit was selected as the experimental subject to isolate the spleen.

[0071] 2. Spleen Cell Isolation

[0072] The spleen of No. 2 New Zealand white rabbits was placed in DMEM basal culture medium containing 100 U / mL penicillin and 100 μg / mL streptomycin, minced into pieces with a scalpel blade, and then transferred to a 100 μm cell sieve for grinding.

[0073] The obtained cell suspension was filtered to remove large cell aggregates and tissue capsules, centrifuged at 450 g for 7 min, and the supernatant was removed to retain the spleen cell aggregates.

[0074] The spleen cell clusters were resuspended in regular PBS buffer and lysed red blood cells, then centrifuged at 450 g for 7 min again, and the spleen cells were reserved.

[0075] The spleen cells were resuspended in DMEM base medium containing 100 U / mL penicillin and 100 μg / mL streptomycin, centrifuged at 450 g for 7 min, and the obtained spleen cells were resuspended in complete medium (DMEM base medium containing 15% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin) for standby.

[0076] 3. B lymphocyte sorting

[0077] The specific sorting method of B lymphocytes can refer to the method disclosed in the authorized Chinese patent CN110016462B "Method for efficiently separating single antigen-specific B lymphocytes from spleen cells". As shown in the figure, the R5 region in the figure is the sorted B lymphocytes, which are cultured in a 96-well cell culture plate, with 1 B lymphocyte per well. Figure 3

[0078] 4. Cloning of rabbit monoclonal antibody gene

[0079] The supernatant of the cultured B cells was identified by antigen-coated ELISA, and the positive control was prepared by diluting the rabbit serum after immunization at 1:2000, and the negative control was PBS buffer. As shown in Table 1.

[0080] Table 1. Elisa identification results of B cell supernatant after culture (OD 450 - OD 620 )

[0081]

[0082] In Table 1, the serial numbers in the first row and the first column refer to the number of the 96-well enzyme-labeled plate; the data with bold and underlined font are the selected positive clones for the next step. After collecting and lysing the cells of the positive clones, RNA was extracted and reverse transcribed into cDNA. Using cDNA as a template, under the action of DNA polymerase, through denaturation, annealing, extension and amplification, the naturally paired rabbit monoclonal antibody light and heavy chain variable region genes (VH and VL) were amplified and sequenced.

[0083] 5. Production and purification of monoclonal antibodies

[0084] ​The rabbit monoclonal antibodies (i.e., B-5, C-9, D-3, D-7, F-1 and F-7) corresponding to the bold and underlined data in Table 1 above were taken as objects, in order to obtain multiple strains of rabbit monoclonal antibodies recognizing dupilumab, the heavy chain and light chain genes of the rabbit monoclonal antibodies were respectively loaded on expression vectors, and the plasmids were transfected into 293F cells; after 72-96 h of transfection, the culture supernatant containing the recombinant rabbit monoclonal antibodies recognizing dupilumab was obtained.

[0085] The recombinant rabbit monoclonal antibodies recognizing dupilumab were purified from the culture supernatant after transfection using protein G affinity gel resin, and the antibodies were identified and then aliquoted and stored at -20°C for standby use. The six strains in Table 1 were subjected to final sequencing, four strains failed sequencing and were discarded (cell death during culture, or loss of the ability to secrete antibodies, etc.), and the remaining two strains of recombinant rabbit monoclonal antibodies were subjected to gel electrophoresis as shown in Figure 3 From Dupilumab it can be seen that the rabbit monoclonal recombinant antibody bands are uniform, the molecular weight is correct, and the antibodies are successfully expressed.

[0086] Example 2: Screening and identification of antibodies

[0087] The two strains of recombinant rabbit monoclonal antibodies screened in Example 1 were subjected to indirect ELISA titer detection by indirect ELISA experiment. The specific method for detection was as follows:

[0088] (1) Dupilumab (DUPILUMAB) was coated using carbonate buffer (pH=9.6, 0.05 M), the coating concentration was 2 μg / mL, 100 μL / well, and the plate was incubated at 4°C overnight; the washing solution was used to wash the plate. Dupilumab

[0089] (2) Blocking was performed using phosphate buffer (pH=7.2, 0.05 M) containing 3% bovine serum albumin and 0.05% Tween-20, 300 μL / well.

[0090] (3) The plate was shaken into the water pool, and 1×PBST was used for washing 3 times, 300 μL / well, and then the plate was patted dry.

[0091] (4) The two strains of recombinant rabbit monoclonal antibodies were sequentially subjected to dilution operation to obtain a total of 7 standard points of 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, 0.0625 μg / mL, 0.03125 μg / mL and 0.015625 μg / mL, and finally 100 μL of phosphate buffer was added as a zero standard (i.e., without standard), and was added to the enzyme-labeled plate, a total of 8 points, and then incubated at 37°C for 1 h, and then the plate was washed with the washing solution.

[0092] ​(5) Pour the primary antibody in the plate into the water pool, wash three times with 1×PBST, 300 μL / well, and pat dry.

[0093] (6) Add horseradish peroxidase-labeled goat anti-rabbit secondary antibody (Jackson Immunoresearch, catalog number 111-035-045) diluted in phosphate buffer; the working concentration of the secondary antibody is 0.08 μg / mL, 100 μL / well, and incubate at 37°C for 30 min.

[0094] (7) Washing the plate: Dump the secondary antibody in the plate into the water pool, wash three times with 1×PBST, 300 μL / well, and pat dry.

[0095] (8) Color development: Add TMB at 100 μL / well and incubate at 37°C for 10 min.

[0096] (9) Termination and Reading: Add 50 μL / well of 2 M HCl to terminate the reaction and read immediately. Read the sample at a wavelength of 450-620 nm on a microplate reader, export the data, and save it. The test results are shown in Table 2 below.

[0097] Table 2 Elisa results of recombinant rabbit monoclonal antibody (OD 450 and OD 620 The difference between

[0098]

[0099] The Biacore 8000 biomolecular interaction analyzer was used to analyze the Dupilumab obtained in this experiment. Dupilumab The affinity of the selected primary and secondary antibodies was precisely determined using rabbit monoclonal antibodies. The selected primary and secondary antibodies were each used at a concentration of 50 nM and immobilized on a CM5 chip (GE Healthcare).

[0100] Then, for the primary antibody, five concentrations of dupilumab (50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM) were used. Figure 4 ) to bind and obtain the affinity curve, such as Dupilumab shown.

[0101] For the secondary antibody, five concentrations of dupilumab (50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.125 nM) were used. Figure 5 ) to bind and obtain the affinity curve, such as Figure 4 shown.

[0102] Figures 4-5 、 5In the figure, the horizontal axis is the reaction time, the vertical axis is the response value, and the colored lines are the response values ​​under different concentrations of antibody loading. The binding constant Ka and dissociation constant Kd are obtained based on the response value. The affinity value KD is calculated according to the formula KD=Kd / Ka. The smaller the KD value, the stronger the antibody affinity. Generally, the antibody affinity is less than 10 -10 The affinity of the antibody is considered to be very high. Tables 3 and 4 below are the affinity test results of the first antibody and the second antibody respectively.

[0103] Table 3 Affinity test results of the first antibody

[0104]

[0105] Table 4 Affinity test results of the second antibody

[0106]

[0107] From Table 3-4 and Dupilumab It can be seen that the affinity of the first antibody of Dupilumab rabbit monoclonal antibody was finally obtained by curve fitting and calculation to be 7.36×10 -11 M; the affinity of the secondary antibody of dupilumab rabbit monoclonal antibody is 9.44×10 -11 M.

[0108] The complete sequences of the heavy and light chains of the first and second antibodies were obtained by outsourcing sequencing to a third-party company (Qingke Bio). Then, according to the Kabat rules (reference: Kabat EA, Wu TT, Bilofsky H. Attempts to locate residues incomplementarity-determining regions of antibody combining sites that make contact with antigen. Proc. Natl. Acad. Sci. USA. 1976; 73: 617–619), the sequences of the complementarity-determining regions (CDR1, CDR2, and CDR3) of the heavy and light chains of the two antibodies (i.e., the first and second antibodies) were obtained.

[0109] The anti-duplimab obtained in this example ( Dupilumab ) The amino acid sequence of the heavy chain of the first antibody of the rabbit monoclonal antibody is:

[0110] QSVEESGGRLVTPGTPLTLTCKASGFSLSRYWMSWVRQAPGEGLEWIGYISSGGSSYYATWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARDAGYTIGTPLNIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK, as set forth in SEQ ID NO. 8.

[0111] The light chain amino acid sequence of the first antibody is:

[0112] DVVMTQTPASVSEPVGDTVTIKCQASEDIESYLAWYQQKPGQPPKLLIYGASNLESGVPSRFKGSGSGTEYTLTISDLECADAATYYCQCTYYPDNAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC, as set forth in SEQ ID NO. 9.

[0113] wherein the heavy chain variable region of the first antibody has the amino acid sequence:

[0114] QSVEESGGRLVTPGTPLTLTCKASGFSLSRYWMSWVRQAPGEGLEWIGYISSGGSSYYATWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARDAGYTIGTPLNIWGPGTLVTVSS, as set forth in SEQ ID NO. 6.

[0115] The light chain variable region of the first antibody has the amino acid sequence:

[0116] the amino acid sequence of the complementarity determining region CDR1 on the heavy chain of the first antibody is GFSLSRYW, as shown in SEQ ID NO. 1;

[0117] the amino acid sequence of the complementarity determining region CDR1 on the heavy chain of the first antibody is GFSLSRYW, as shown in SEQ ID NO. 1;

[0118] the amino acid sequence of the complementarity determining region CDR2 on the heavy chain of the first antibody is ISSGGSS, as shown in SEQ ID NO. 2;

[0119] the amino acid sequence of the complementarity determining region CDR3 on the heavy chain of the first antibody is ARDAGYTIGTPLNI, as shown in SEQ ID NO. 3;

[0120] the amino acid sequence of the complementarity determining region CDR1 on the heavy chain of the first antibody is GFSLSRYW, as shown in SEQ ID NO. 1;

[0121] the amino acid sequence of the complementarity determining region CDR2 on the heavy chain of the first antibody is ISSGGSS, as shown in SEQ ID NO. 2;

[0122] the amino acid sequence of the complementarity determining region CDR3 on the heavy chain of the first antibody is ARDAGYTIGTPLNI, as shown in SEQ ID NO. 3;

[0123] the amino acid sequence of the complementarity determining region CDR1 on the heavy chain of the first antibody is GFSLSRYW, as shown in SEQ ID NO. 1; Dupilumab the amino acid sequence of the complementarity determining region CDR1 on the heavy chain of the first antibody is GFSLSRYW, as shown in SEQ ID NO. 1;

[0124] QSVEESGGRLVTPGTPLTLTCTVSGIDLSSYAMSWVRQAPGKGLEWIGIISISGSTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARYDTYDDYGDYDGFNLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK, as set forth in SEQ ID NO. 17.

[0125] The light chain amino acid sequence of the second antibody is:

[0126] ALVMTQTPASVEAAVGGTVTIKCQASQSIYSNLAWYQQKPGQPPNLLIYAASYLASGVSSRFKGSGSGTQFTLTISDLECADAATYYCQQDYTNNDVDNTFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC, as set forth in SEQ ID NO. 18.

[0127] wherein the amino acid sequence of the heavy chain variable region of the second antibody is:

[0128] QSVEESGGRLVTPGTPLTLTCTVSGIDLSSYAMSWVRQAPGKGLEWIGIISISGSTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARYDTYDDYGDYDGFNLWGPGTLVTVSS, as set forth in SEQ ID NO. 15.

[0129] The amino acid sequence of the light chain variable region of the second antibody is:

[0130] The amino acid sequence of the light chain variable region of the second antibody is:

[0131] The amino acid sequence of the light chain variable region of the second antibody is:

[0132] The amino acid sequence of the light chain variable region of the second antibody is:

[0133] The amino acid sequence of the light chain variable region of the second antibody is:

[0134] The amino acid sequence of the light chain variable region of the second antibody is:

[0135] The amino acid sequence of the light chain variable region of the second antibody is:

[0136] The amino acid sequence of the light chain variable region of the second antibody is:

[0137] Example 3: Anti-dupilumab (Dupixent®) rabbit monoclonal antibody first antibody and second antibody sensitivity detection Dupilumab Example 4: Anti-dupilumab (Dupixent®) rabbit monoclonal antibody first antibody and second antibody specificity detection

[0138] Example 5: Anti-dupilumab (Dupixent®) rabbit monoclonal antibody first antibody and second antibody cross-reactivity detection Dupilumab Example 6: Anti-dupilumab (Dupixent®) rabbit monoclonal antibody first antibody and second antibody detection of human serum samples

[0139] 1. Recombinant protein IL4R was coated with carbonate buffer (pH = 9.6, 0.05 M) at a concentration of 1 μg / mL, 100 μL / well, 4°C overnight; wash the plate with washing solution.

[0140] 2. Blocking with phosphate buffer (pH = 7.2, 0.05 M) containing 3% bovine serum albumin and 0.05% Tween-20.

[0141] 3. Anti-dupilumab (Dupixent®) rabbit monoclonal antibody first antibody and second antibodyFigure 6 The first antibody and the second antibody of the rabbit monoclonal antibody were diluted with phosphate buffer to obtain a total of 7 standard points 800 ng / mL, 400 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 25 ng / mL, and 12.5 ng / mL, respectively, and then phosphate buffer was added as a zero standard to obtain a total of 8 standard points, and 50 μL of each was added to the enzyme-labeled plate. Dupilumab was diluted with phosphate buffer to 100 ng / mL, 50 μL of each was added to the enzyme-labeled plate, mixed well, and incubated at 37°C for 1 h, and then the plate was washed with washing solution.

[0142] 4, The horseradish peroxidase-labeled goat anti-rabbit secondary antibody (jackson immunoresearch, item number 111-035-045) diluted with phosphate buffer was added; the working concentration of the secondary antibody was 0.08 μg / mL, and the plate was incubated at 37°C for 0.5 h, and then the plate was washed with washing solution.

[0143] 5, TMB color developing solution was added, and color development was performed at room temperature for 10 min; 50 μL of 2 M hydrochloric acid was added to terminate color development, and then the absorbance values were measured at wavelengths of 450 nm and 630 nm, respectively, and OD 450 The OD 630 was subtracted, and the corrected absorbance value was used for inhibition rate calculation. The inhibition rate calculation formula was: inhibition rate = (1- sample or standard OD value / blank control OD value) x 100%.

[0144] 6, The standard curve was plotted with the logarithmic value of the concentration of the anti-Dupilumab rabbit monoclonal antibody as the abscissa and the inhibition rate as the ordinate. The curve is shown in Figure 7 The experimental results show that the competitive enzyme-linked immunoassay method established based on the first antibody and the second antibody of the anti-Dupilumab rabbit monoclonal antibody, the first antibody is a competitive antibody, and the IC50 is 64.59 ng / mL, and the second antibody is a non-competitive antibody.

[0145] Example 4: Performance detection of the first antibody and the second antibody of the anti-Dupilumab rabbit monoclonal antibody

[0146] 1, Specificity detection

[0147] Human IgG4 and human total IgG consistent with the subtype of Dupilumab were taken for the detection of the specificity of the first antibody and the second antibody of the anti-Dupilumab rabbit monoclonal antibody.

[0148] An indirect enzyme-linked immunosorbent assay (ELISA) was used. Dupilumab, human IgG4, and total human IgG were coated on the surface of the sample. The primary and secondary antibodies were used as detection antibodies. The coating concentrations of dupilumab, human IgG4, and total human IgG were all 1 μg / mL.

[0149] like Figure 8 As shown, in the indirect enzyme-linked immunosorbent assay (ELISA) method using the primary and secondary antibodies provided by the present invention, the primary and secondary antibodies, both of which are rabbit monoclonal antibodies against dupilumab, showed no cross-reactivity with either human IgG4 or total human IgG. This demonstrates that the primary and secondary antibodies provided by the present invention are highly specific for dupilumab.

[0150] 2. Thermal stability test

[0151] Dupilumab rabbit monoclonal primary and secondary antibodies were placed in a 37°C incubator and sampled on day 14. Dupilumab was detected using an indirect enzyme-linked immunosorbent assay (ELISA). Untreated primary and secondary antibodies (i.e., primary and secondary antibodies, respectively, were stored at -20°C) served as controls. Reaction curves were generated for the antibody samples treated at 37°C for 14 days and the untreated control samples.

[0152] like ​ 、 9 As shown, after treatment at 37°C for 14 days, the anti-dupilumab rabbit monoclonal primary and secondary antibodies provided by the present invention showed no obvious precipitation or deterioration, no significant decrease in concentration, and no significant decrease in indirect enzyme-linked immunosorbent assay signal, with the results showing a signal reduction of less than 5%. This demonstrates that the anti-dupilumab rabbit monoclonal primary and secondary antibodies provided by the present invention have strong thermal stability.

[0153] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. A high-affinity rabbit monoclonal antibody against dupilumab, characterized in that: The high-affinity rabbit monoclonal antibody is the first antibody or the second antibody; The amino acid sequence of the complementarity determining region CDR1 on the heavy chain of the first antibody is shown in SEQ ID NO.1; The amino acid sequence of the complementarity determining region CDR2 on the heavy chain of the first antibody is shown in SEQ ID NO.2; The amino acid sequence of the complementarity determining region CDR3 on the heavy chain of the first antibody is shown in SEQ ID NO.3; The amino acid sequence of the complementarity determining region CDR1 on the light chain of the first antibody is shown in SEQ ID NO.4; The amino acid sequence of the complementarity determining region CDR2 on the light chain of the first antibody is GAS; The amino acid sequence of the complementarity determining region CDR3 on the light chain of the first antibody is shown in SEQ ID NO.5; The amino acid sequence of the complementarity determining region CDR1 on the heavy chain of the second antibody is shown in SEQ ID NO.10; The amino acid sequence of the complementarity determining region CDR2 on the heavy chain of the second antibody is shown in SEQ ID NO.11; The amino acid sequence of the complementarity determining region CDR3 on the heavy chain of the second antibody is shown in SEQ ID NO.12; The amino acid sequence of the complementarity determining region CDR1 on the light chain of the second antibody is shown in SEQ ID NO.13; The amino acid sequence of the complementarity determining region CDR2 on the light chain of the second antibody is GAS; The amino acid sequence of the complementarity determining region CDR3 on the light chain of the second antibody is shown in SEQ ID NO.

14.

2. The high-affinity rabbit monoclonal antibody against dupilumab according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the first antibody is shown in SEQ ID NO.6; the amino acid sequence of the light chain variable region of the first antibody is shown in SEQ ID NO.7; The amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the second antibody is shown in SEQ ID NO.

16.

3. The high-affinity rabbit monoclonal antibody against dupilumab according to claim 2, characterized in that The amino acid sequence of the heavy chain of the first antibody is shown in SEQ ID NO.8; the amino acid sequence of the light chain of the first antibody is shown in SEQ ID NO.9; The amino acid sequence of the heavy chain of the second antibody is shown in SEQ ID NO.17; the amino acid sequence of the light chain of the second antibody is shown in SEQ ID NO.

18.

4. A substance, characterized in that The substance includes any one of the following aspects: (1) A nucleic acid molecule or a vector comprising the nucleic acid molecule, wherein the nucleic acid encodes the high-affinity rabbit monoclonal antibody according to any one of claims 1 to 3; (2) A modified cell or recombinant strain, wherein the modified cell or recombinant strain is used to express the high-affinity rabbit monoclonal antibody according to any one of claims 1 to 3; (3) An antibody derivative comprising the high-affinity rabbit monoclonal antibody according to any one of claims 1 to 3, directly or indirectly linked to a detectable marker, drug or toxin; (4) A detection product comprising the high-affinity rabbit monoclonal antibody according to any one of claims 1 to 3, or the nucleic acid molecule, or the vector, or a derivative of the high-affinity rabbit monoclonal antibody.

5. The substance according to claim 4, characterized in that The detection product is a competitive ELISA detection kit for detecting dupilumab.

6. The substance according to claim 5, characterized in that In the competitive ELISA detection kit for detecting dupilumab, the first antibody is a competitive antibody and the second antibody is a non-competitive antibody.

7. Use of the high-affinity rabbit monoclonal antibody against dupilumab according to any one of claims 1 to 3, characterized in that: Used to detect dupilumab for purposes other than disease diagnosis and treatment; or, used to prepare products for detecting dupilumab.

8. The use of the high-affinity rabbit monoclonal antibody against dupilumab according to claim 7, characterized in that: The product is a detection reagent, a detection kit, a detection test strip, a detection probe or a detection chip.

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