Monoclonal antibody for identifying TNFalpha-CRM197 vaccine and preparation method thereof

Abstract

The invention provides a monoclonal antibody for recognizing a TNF (Tumor Necrosis Factor) alpha-CRM197 vaccine and a preparation method of the monoclonal antibody. The affinity of the monoclonal antibody prepared by screening to a TNF alpha-CRM197 vaccine is higher than that of TNF alpha, the monoclonal antibody can be used for specifically detecting the relative antigen amount (in vitro relative titer) in the TNF alpha-CRM197 vaccine after being combined with adalimumab, and the monoclonal antibody hardly responds to free TNF alpha.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a monoclonal antibody for recognizing TNFα-CRM197 vaccine and a preparation method thereof. BACKGROUND

[0002] TNFα is a key factor in the pathogenesis of autoimmune diseases such as rheumatoid arthritis and ankylosing spondylitis. In the past one or two decades, at least three types of TNFα antagonists (inhibitors) have been marketed, and through clinical trials and real-world clinical applications, it has been confirmed that TNFα inhibitors can reduce the inflammatory response of RA joints, improve joint function, and delay the progression of the disease. The application of TNFα antagonists in the treatment of RA has been widely recognized and applied, and has achieved remarkable results, and has attracted high attention in the field of medical research and development. However, TNFα antagonists including Enbrel and Simponi have the characteristics of large dosage, high frequency of medication, and high manufacturing cost, therefore, there is an urgent clinical and market demand for developing a new type of, economical, more durable, and more efficient drug that antagonizes TNFα.

[0003] TNFα-CRM197 therapeutic vaccine (hereinafter referred to as "TNFα-CRM197 vaccine") is a new type of combined vaccine formed by cross-linking TNFα and carrier protein CRM197. The main mechanism of the vaccine is to induce the production of high levels of anti-TNFα antibodies in the body, neutralize TNFα in the body, inhibit the TNFα / TNFR signaling pathway, and then inhibit the production of inflammatory factors, so as to achieve the purpose of treating diseases such as rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, and Crohn's disease.

[0004] TNFα-CRM197 vaccine is a product of non-directional coupling of TNFα and carrier protein, therefore, the purification and antigen content determination of TNFα-CRM197 vaccine are key process steps and quality control methods. The purpose of the present application is to screen an antibody that can specifically recognize TNFα in TNFα-CRM197 vaccine, and to use a double-antibody sandwich method to specifically recognize TNFα in TNFα-CRM197 vaccine, so as to solve the key problems in the quality control of TNFα-CRM197 vaccine. SUMMARY

[0005] The purpose of the present application is to provide a monoclonal antibody for recognizing TNFα in TNFα-CRM197 vaccine and its application. The monoclonal antibody can bind to TNFα-CRM197 vaccine, and in the antigen content determination of TNFα-CRM197 vaccine, the combination of adalimumab can specifically recognize TNFα antigen in the conjugate, and almost does not respond to free TNFα, that is, it can be applied to the determination of the relative antigen amount (in vitro relative titer) of TNFα-CRM197 vaccine.

[0006] In a first aspect of the application, there is provided a monoclonal antibody or an antigen-binding fragment thereof, which recognizes a TNFa antigen in a TNFa-CRM197 vaccine, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the amino acid sequences of the complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3 in the heavy chain variable region (VH) are the amino acid sequences of VH-CDR1, VH-CDR2 and VH-CDR3 in the heavy chain variable region (VH) as set forth in SEQ ID NO: 1, respectively, and the amino acid sequences of the complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3 in the light chain variable region (VL) are the amino acid sequences of VL-CDR1, VL-CDR2 and VL-CDR3 in the light chain variable region (VL) as set forth in SEQ ID NO: 9, respectively.

[0007] In another preferred embodiment, the rules for the division of the complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3 in the heavy chain variable region (VH) and the complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3 in the light chain variable region (VL) are Kabat, AbM, Chothia, Contact or IMGT.

[0008] In another preferred embodiment, the heavy chain variable region (VH) of the antibody or antigen-binding fragment thereof has the following complementarity determining regions CDRs: VH-CDR1 as set forth in SEQ ID NO: 2, VH-CDR2 as set forth in SEQ ID NO: 3, and VH-CDR3 as set forth in SEQ ID NO: 4; The light chain variable region (VL) of the antibody or antigen-binding fragment thereof has the following complementarity determining regions CDRs: VL-CDR1 as set forth in SEQ ID NO: 10, VL-CDR2 as set forth in SEQ ID NO: 11, and VL-CDR3 as set forth in SEQ ID NO: 12; wherein any of the above amino acid sequences further comprises a derivative sequence obtained by optionally adding, deleting, modifying and / or substituting at least one (e.g., 1-3) amino acid.

[0009] In another preferred embodiment, the derivative sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any of the above amino acid sequences.

[0010] In another preferred embodiment, the VH-CDR1, VH-CDR2, and VH-CDR3 are separated by framework regions VH-FR1, VH-FR2, VH-FR3, and VH-FR4.

[0011] In another preferred embodiment, the amino acid sequence of the VH-FR1 is set forth in SEQ ID NO: 5, the amino acid sequence of the VH-FR2 is set forth in SEQ ID NO: 6, the amino acid sequence of the VH-FR3 is set forth in SEQ ID NO: 7, and the amino acid sequence of the VH-FR4 is set forth in SEQ ID NO: 8.

[0012] In another preferred embodiment, the VL-CDR1, VL-CDR2, and VL-CDR3 are separated by framework regions VL-FR1, VL-FR2, VL-FR3, and VL-FR4.

[0013] In another preferred embodiment, the amino acid sequence of the VL-FR1 is set forth in SEQ ID NO: 13, the amino acid sequence of the VL-FR2 is set forth in SEQ ID NO: 14, the amino acid sequence of the VL-FR3 is set forth in SEQ ID NO: 15, and the amino acid sequence of the VL-FR4 is set forth in SEQ ID NO: 16.

[0014] In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is SEQ ID NO: 1.

[0015] In another preferred embodiment, the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof is SEQ ID NO: 9.

[0016] In another preferred embodiment, the antibody or antigen-binding fragment thereof comprises the VL and VH sequences set forth in Table B.

[0017] In another preferred embodiment, the antibody or antigen-binding fragment thereof comprises the CDR and FR sequences set forth in Table B.

[0018] In another preferred embodiment, the antibody or antigen-binding fragment thereof comprises a monomer, a bivalent antibody, and / or a multivalent antibody.

[0019] In another preferred embodiment, the bivalent antibody is a bispecific antibody.

[0020] In another preferred embodiment, the multivalent antibody is a multispecific antibody.

[0021] In another preferred embodiment, the antibody or antigen-binding fragment thereof comprises a murine antibody, a humanized antibody, or a chimeric antibody.

[0022] In another preferred embodiment, the antibody or antigen-binding fragment thereof is a partially or fully humanized, or fully human monoclonal antibody.

[0023] In another preferred embodiment, the antigen-binding fragment is selected from the group consisting of scFv, Fab, Fab', F(ab')2, Fv fragment, heavy chain antibody, disulfide-bonded Fv (dsFv).

[0024] In another preferred embodiment, the heavy chain constant region of the antibody or antigen-binding fragment thereof is selected from the group consisting of human IgGl, IgG2, IgG3, or IgG4.

[0025] In another preferred embodiment, the light chain constant region of the antibody or antigen-binding fragment thereof is selected from the group consisting of constant region of human antibody kappa chain or lambda chain.

[0026] In a second aspect of the present application, there is provided a recombinant protein having: (i) the antibody or antigen-binding fragment thereof as described in the first aspect of the present application; and (ii) an optional tag sequence to assist expression and / or purification.

[0027] In another preferred embodiment, the tag sequence comprises Fc tag, HA tag, GGGS sequence, FLAG tag, Myc tag, 6His tag, or a combination thereof.

[0028] In another preferred embodiment, the recombinant protein comprises a fusion protein.

[0029] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a multimer.

[0030] In a third aspect of the present application, there is provided a polynucleotide encoding the antibody or antigen-binding fragment thereof as described in the first aspect of the present application, or the recombinant protein as described in the second aspect of the present application.

[0031] In another preferred embodiment, the polynucleotide is RNA (including mRNA), DNA (including cDNA).

[0032] In another preferred embodiment, the polynucleotide comprises: a heavy chain variable region encoding sequence as set forth in SEQ ID NO: 17, and a light chain variable region encoding sequence as set forth in SEQ ID NO: 18.

[0033] In a fourth aspect of the present application, there is provided a vector containing the polynucleotide as described in the third aspect of the present application.

[0034] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, viral vector, plasmid, transposon, other gene transfer system, or a combination thereof.

[0035] In another preferred embodiment, the vector comprises a viral vector, such as lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

[0036] In another preferred embodiment, the vector is selected from the group consisting of pTomo lentivirus vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV, etc.

[0037] In another preferred embodiment, the vector further comprises an element selected from the group consisting of a promoter, a transcription enhancer element WPRE, a long terminal repeat sequence LTR, etc.

[0038] In a fifth aspect of the present application, a host cell is provided, which comprises the vector as described in the fourth aspect of the present application, or has integrated into its genome the polynucleotide as described in the third aspect of the present application.

[0039] In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.

[0040] In another preferred embodiment, the host cell is selected from the group consisting of E. coli, a yeast cell, a mammalian cell.

[0041] In a sixth aspect of the present application, a detection kit is provided, which comprises: the antibody or antigen binding fragment thereof as described in the first aspect of the present application or the recombinant protein as described in the second aspect of the present application.

[0042] In another preferred embodiment, the detection kit further comprises: adalimumab.

[0043] In another preferred embodiment, the detection kit comprises: (a1) the antibody or antigen binding fragment thereof as described in the first aspect of the present application or the recombinant protein as described in the second aspect of the present application as a coating antibody; (b1) adalimumab as a detection antibody.

[0044] In another preferred embodiment, the detection kit comprises: (a2) adalimumab as a coating antibody; (b2) the antibody or antigen binding fragment thereof as described in the first aspect of the present application or the recombinant protein as described in the second aspect of the present application as a detection antibody.

[0045] In another preferred embodiment, the adalimumab is enzyme-labeled adalimumab.

[0046] In another preferred embodiment, the enzyme-labeled adalimumab is: adalimumab conjugated with horseradish peroxidase (HRP), or adalimumab conjugated with alkaline phosphatase (ALP).

[0047] In another preferred embodiment, the antibody or antigen-binding fragment thereof according to the first aspect of the present application or the recombinant protein according to the second aspect of the present application is enzyme-labeled.

[0048] In another preferred embodiment, the enzyme label comprises: horseradish peroxidase (HRP) label, alkaline phosphatase (ALP) label.

[0049] In another preferred embodiment, the detection kit is a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit.

[0050] In another preferred embodiment, the detection kit further comprises: a TNFα-CRM197 conjugate or a TNFα-CRM197 vaccine as a standard.

[0051] In another preferred embodiment, the TNFα-CRM197 conjugate or the TNFα-CRM197 vaccine is a series of dilutions of the standard, and the concentration of the standard comprises: 160 μg / ml, 80 μg / ml, 40 μg / ml, 20 μg / ml, 10 μg / ml, 5 μg / ml, 2.5 μg / ml, 1.25 μg / ml, 0.625 μg / ml, 0.3125 μg / ml, 0.15625 μg / ml, 0.078125 μg / ml.

[0052] In another preferred embodiment, the detection kit further comprises: a color developing solution (i.e. an enzyme substrate solution, such as TMB for HRP or PNPP for ALP), a termination solution (such as H2SO4 for HRP reaction or NaOH solution for ALP reaction), a sample diluent, a positive control, a negative control, or a combination thereof.

[0053] In a seventh aspect of the present application, there is provided a method of producing a recombinant polypeptide, which is an antibody or antigen-binding fragment thereof according to the first aspect of the present application or a recombinant protein according to the second aspect of the present application, the method comprising the steps of: (a) culturing the host cell according to the fifth aspect of the present application under conditions suitable for expression, thereby obtaining a culture comprising the recombinant polypeptide; and (b) isolating and / or purifying the recombinant polypeptide from the culture.

[0054] In an eighth aspect of the present application, there is provided a use of the antibody or antigen binding fragment thereof according to the first aspect of the present application, the recombinant protein according to the second aspect of the present application, the polynucleotide according to the third aspect of the present application, the vector according to the fourth aspect of the present application, or the host cell according to the fifth aspect of the present application, in the preparation of a detection reagent or a detection kit.

[0055] In another preferred embodiment, the detection kit is the detection kit according to the sixth aspect of the present application.

[0056] In another preferred embodiment, the detection reagent or the detection kit is used for detecting the content of the TNFa antigen in the TNFa-CRM197 vaccine.

[0057] In a ninth aspect of the present application, there is provided a detection method for detecting the content of the TNFa antigen in the TNFa-CRM197 vaccine, the detection method comprising the steps of: (S1) providing a sample to be detected; (S2) combining the antibody or antigen binding fragment thereof according to the first aspect of the present application or the recombinant protein according to the second aspect of the present application with adalimumab into an antibody pair, and performing a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) on the sample to be detected, thereby detecting the content of the TNFa antigen in the TNFa-CRM197 vaccine.

[0058] In another preferred embodiment, the detection method further comprises the step of providing the detection kit according to the sixth aspect of the present application.

[0059] In another preferred embodiment, in the antibody pair, the antibody or antigen binding fragment thereof according to the first aspect of the present application or the recombinant protein according to the second aspect of the present application is used as a coating antibody, and adalimumab is used as a detection antibody.

[0060] In another preferred embodiment, in the antibody pair, adalimumab is used as a coating antibody, and the antibody or antigen binding fragment thereof according to the first aspect of the present application or the recombinant protein according to the second aspect of the present application is used as a detection antibody.

[0061] In another preferred embodiment, the adalimumab is enzyme-labeled adalimumab.

[0062] In another preferred embodiment, the enzyme-labeled adalimumab is horseradish peroxidase (HRP)-coupled adalimumab or alkaline phosphatase (ALP)-coupled adalimumab.

[0063] In another preferred embodiment, the antibody or antigen binding fragment thereof according to the first aspect of the present application or the recombinant protein according to the second aspect of the present application is enzyme-labeled.

[0064] In another preferred embodiment, the enzyme label comprises: horseradish peroxidase (HRP) label, alkaline phosphatase (ALP) label.

[0065] In another preferred embodiment, the sample to be detected comprises: one or more TNFα-CRM197 vaccines.

[0066] In another preferred embodiment, the detection method comprises the steps of: Step 1: coating the antibody or antigen-binding fragment thereof as described in the first aspect of the present application or the recombinant protein as described in the second aspect of the present application on an enzyme-labeled plate at a certain concentration; Step 2: after washing the enzyme-labeled plate, the enzyme-labeled plate is blocked with a blocking solution; Step 3: after washing the enzyme-labeled plate, the sample to be detected and the TNFα-CRM197 conjugate or TNFα-CRM197 vaccine standard are respectively diluted by a certain multiple and incubated on the enzyme-labeled plate; Step 4: after washing the enzyme-labeled plate, HRP-labeled adalimumab is added to each reaction well, and incubated at room temperature for 1 hour; Step 5: after washing the enzyme-labeled plate, color developing solution is added for color development, and then stop solution is added to stop the reaction; Step 6: measuring the absorbance at 450 nm; Step 7: according to the measured absorbance, the content of TNFα antigen in the sample to be detected is calculated by combining the standard curve equation.

[0067] In another preferred embodiment, the TNFα-CRM197 conjugate or TNFα-CRM197 vaccine is a series of diluted standards, and the concentration of the standard comprises: 160 μg / ml, 80 μg / ml, 40 μg / ml, 20 μg / ml, 10 μg / ml, 5 μg / ml, 2.5 μg / ml, 1.25 μg / ml, 0.625 μg / ml, 0.3125 μg / ml, 0.15625 μg / ml, 0.078125 μg / ml.

[0068] In another preferred embodiment, the color developing solution is TMB.

[0069] In another preferred embodiment, the stop solution is H2SO4.

[0070] In another preferred embodiment, the detection method further comprises the step of: taking the protein concentration (μg / ml) of the TNFα-CRM197 conjugate or TNFα-CRM197 vaccine standard as the abscissa, taking the OD450 average value as the ordinate, fitting a four-parameter logistic curve, and obtaining the standard curve equation.

[0071] In the tenth aspect of the present invention, a method for detecting the content of TNFα antigen in TNFα-CRM197 vaccine is provided, which comprises the use of the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, the recombinant protein as described in the second aspect of the present invention, and the detection kit as described in the sixth aspect of the present invention.

[0072] In another preferred embodiment, the detection method is the detection method described in the ninth aspect of the present invention.

[0073] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 Shown are the Western blot results of hybridoma cell culture supernatant for detection of TNFα.

[0075] Figure 2 The results of ELISA for detecting TNFα using culture supernatants of monoclonal hybridoma cells are shown.

[0076] Figure 3 The ELISA results of detecting TNFα-CRM197 vaccine using the culture supernatant of monoclonal hybridoma cells are shown.

[0077] Figure 4 The dose-response curves of the relative antigen amounts of three batches of TNFα-CRM197 vaccine detected by double-antibody sandwich ELISA using a combination of monoclonal antibody 21M2 and adalimumab are shown. DETAILED DESCRIPTION

[0078] The present inventors have developed a monoclonal antibody having higher affinity to TNFα-CRM197 vaccine than TNFα through extensive and intensive research, and a large number of screenings. Specifically, the antibody of the present application has complementarity determining regions VH-CDR1, VH-CDR2, and VH-CDR3 contained in the heavy chain variable region (VH) shown in SEQ ID NO: 1, and has complementarity determining regions VL-CDR1, VL-CDR2, and VL-CDR3 contained in the light chain variable region (VL) shown in SEQ ID NO: 9. Preferably, the antibody of the present application includes each of the VL and VH sequences shown in Table B, as well as the CDR and FR sequences. The antibody of the present application, when combined with adalimumab, establishes a double antibody sandwich ELISA method for specifically detecting the TNFα antigen in the TNFα-CRM197 vaccine, and hardly responds to free TNFα, thereby developing an application for determining the relative antigen amount (in vitro relative titer) of the TNFα-CRM197 vaccine. On this basis, the present application is completed.

[0079] Terminology For the purposes of United States patent practice, the contents of any related application(s) are incorporated by reference in their entirety. In order that the present application can be more readily understood, certain technical and scientific terms are specifically defined herein. Unless specifically defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The present application is not limited to the specific methods and experimental conditions described, since such methods and conditions can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present application will be limited only by the appended claims.

[0080] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value can vary from the recited value by not more than 1%. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0081] The three letter and one letter codes for amino acid residues used herein are in accord with the IUPAC-IUBMB standards as published in J. Biol. Chem., 243, p. 3558 (1968).

[0082] As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance can or can not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of the particular sequence can or can not be present, and can be 1, 2, or 3.

[0083] The term "sequence identity" as used herein refers to the extent to which two nucleic acid or two amino acid sequences are identical, with appropriate mutations such as substitutions, insertions or deletions, when optimally aligned and compared. The sequence identity between a sequence as described herein and a sequence identical thereto can be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

[0084] Antibody As used herein, the term "antibody" or "immunoglobulin" refers to a heterotetrameric glycoprotein of about 150,000 daltons having the same structural characteristics, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. At one end of each heavy chain is a variable region (VH) followed by a number of constant regions. At one end of each light chain is a variable region (VL) followed by a constant region; the constant region of the light chain is aligned with the first constant region of the heavy chain, and the variable region of the light chain is aligned with the variable region of the heavy chain. Particular amino acid residues are located at the interface between the variable regions of the light and heavy chains.

[0085] As used herein, the term "variable" refers to the fact that certain portions of the variable regions differ extensively in sequence among antibodies within a given species, and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable regions of antibodies; it is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable regions. The more highly conserved portions of the variable regions are called the framework regions (FRs). The variable regions of the heavy and light chains each comprise four FR regions, largely

[0086] The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two major types, called kappa and lambda, based on the amino acid sequences of their constant regions. Depending on the amino acid sequences of their heavy chain constant regions, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions of different classes of immunoglobulins are denoted α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally (see, for example, Fundamental Immunology Ch. 1 by Paul, W., ed. Raven Press: New York, 1999).

[0087] Generally, the antigen binding properties of an antibody can be described by three specific regions on the variable regions of the heavy and light chains, called complementarity determining regions (CDRs), which are interspersed with four framework regions (FRs) whose amino acid sequences are relatively conserved and do not directly participate in binding reactions. The CDRs form loops or stretches of amino acid residues and are held in place relative to the FRs by FR β- sheet formation. CDRs on the heavy chain and on the light chain together with their spatial arrangements contribute to the formation of an antigen binding site of an antibody. The identification of the FR and CDR regions can be determined by comparing the amino acid sequences of antibodies of the same class or subclass (see, for example, Kabat, Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1985 and 1991).

[0088] In the present application, "VH-CDR1" and "CDR-H1" are used interchangeably and both refer to CDR1 of the heavy chain variable region; "VH-CDR2" and "CDR-H2" are used interchangeably and both refer to CDR2 of the heavy chain variable region; "VH-CDR3" and "CDR-H3" are used interchangeably and both refer to CDR3 of the heavy chain variable region. "VL-CDR1" and "CDR-L1" are used interchangeably and both refer to CDR1 of the light chain variable region; "VL-CDR2" and "CDR-L2" are used interchangeably and both refer to CDR2 of the light chain variable region; "VL-CDR3" and "CDR-L3" are used interchangeably and both refer to CDR3 of the light chain variable region.

[0089] The present application includes not only intact antibodies, but also fragments of antibodies that are immunologically active or fusion proteins of antibodies with other sequences. Thus, the present application also includes fragments, derivatives, and analogs of the antibodies.

[0090] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes usually consist of chemically reactive groups of atoms on the antigen's surface. These sites can have specific shapes to which an antibody's binding site adapts, or they can have specific chemical properties which interact with functional groups in the antibody. Epitopes are formed from contiguous amino acids or noncontiguous amino acids juxtaposed in three-dimensional space.

[0091] The terms "specifically binds," "selectively binds," "selectively binds," and "specifically binds" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, an antibody will bind to the predetermined antigen with an affinity (KD) of about less than 10 -7 M, for example, about less than 10 -8 M, 10 -9 M, or 10 -10 M or less.

[0092] As used herein, the term "antigenic determinant" refers to a three-dimensional site on an antigen that is recognized by an antibody or antigen binding fragment of the present application.

[0093] In the present application, the antibody has a higher affinity for the TNFα-CRMl97 vaccine than for TNFα, and in combination with adalimumab, specifically detects the TNFα antigen in the TNFα-CRMl97 vaccine, with little or no response to free TNFα.

[0094] In the present application, the antibody includes murine, chimeric, humanized, or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, which include both human and non-human portions, can be obtained by standard DNA recombination techniques, and are useful antibodies. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having a variable region from a murine monoclonal antibody and a constant region from a human immunoglobulin (see, e.g., U.S. Patent 4,816,567 and U.S. Patent 4,816,397, both of which are incorporated herein by reference in their entireties). Humanized antibodies are antibodies derived from non-human species that have one or more complementarity determining regions (CDRs) derived from a non-human species and framework regions derived from a human immunoglobulin molecule (see U.S. Patent 5,585,089, which is incorporated herein by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombination techniques well known in the art.

[0095] In the present application, the antibody can be monospecific, bispecific, trispecific, or more multispecific.

[0096] In the present application, the antibody of the present application also includes conservative variants thereof, which are polypeptides having up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids replaced by similar or identical amino acids as compared to the amino acid sequence of the antibody of the present application. These conservative variant polypeptides are preferably generated by amino acid replacement according to Table A.

[0097] Table A In the present application, the antibody is an antibody that recognizes the TNFα antigen in the TNFα-CRM197 vaccine. The present application provides an antibody having higher affinity to the TNFα-CRM197 vaccine than to TNFα, which comprises a heavy chain containing a heavy chain variable region (VH) amino acid sequence and a light chain containing a light chain variable region (VL) amino acid sequence.

[0098] The heavy chain variable region (VH) has complementarity determining regions (CDRs) selected from the group consisting of: VH-CDR1 as shown in SEQ ID NO: 2, VH-CDR2 as shown in SEQ ID NO: 3, and VH-CDR3 as shown in SEQ ID NO: 4; The light chain variable region (VL) has complementarity determining regions (CDRs) selected from the group consisting of: VL-CDR1 as shown in SEQ ID NO: 10, VL-CDR2 as shown in SEQ ID NO: 11, and VL-CDR3 as shown in SEQ ID NO: 12; wherein any one of the above amino acid sequences further comprises a derivative sequence in which at least one amino acid is optionally added, deleted, modified and / or substituted, and which is capable of retaining higher affinity to the TNFα-CRM197 vaccine than to TNFα.

[0099] In another preferred embodiment, the sequence formed by the addition, deletion, modification and / or substitution of at least one amino acid is preferably an amino acid sequence having at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95% homology or sequence identity.

[0100] Methods to determine sequence homology or identity known to persons of ordinary skill in the art include, but are not limited to, Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). Preferred methods of determining identity are those that result in the greatest match between the sequences tested. Methods of determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG suite of programs (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith Waterman algorithm can also be used to determine identity.

[0101] Preferably, the antibody described herein is one or more of an antibody full-length protein, an antigen antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody fragment (scFv), and a single domain antibody (sdAb), as well as a monoclonal antibody or a polyclonal antibody prepared from the above-mentioned antibodies. The monoclonal antibody can be prepared by various approaches and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc., and the mainstream is to prepare a monoclonal antibody from a wild type or transgenic mouse by hybridoma technology.

[0102] The antibody full-length protein described herein is a conventional antibody full-length protein in the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region. The heavy chain variable region and the light chain variable region of the protein form a full human antibody full-length protein with a human heavy chain constant region and a human light chain constant region. Preferably, the antibody full-length protein is IgG1, IgG2, IgG3, or IgG4; more preferably, IgG1.

[0103] The antibody of the present application can be a double-chain or single-chain antibody, and can be selected from an animal-derived antibody, a chimeric antibody, a humanized antibody, more preferably a humanized antibody, a human-animal chimeric antibody, and more preferably a full humanized antibody.

[0104] The antibody derivative described herein can be a single-chain antibody, and / or an antibody fragment, such as Fab, Fab', F(ab')2, or other known antibody derivatives in the art, etc., as well as any one or several of IgA, IgD, IgE, IgG, and IgM antibodies or other subtypes of antibodies.

[0105] The single-chain antibody described herein is a conventional single-chain antibody in the art, which includes a heavy chain variable region, a light chain variable region, and a short peptide of 15-20 amino acids.

[0106] Preferably, the animal is a mammal, such as a mouse.

[0107] The antibody of the present application can be a chimeric antibody, a humanized antibody, a CDR grafted and / or modified antibody that recognizes the TNFα antigen in the TNFα-CRM197 vaccine.

[0108] In the above description of the present application, the number of added, deleted, modified, and / or substituted amino acids is preferably not more than 40% of the total number of initial amino acids, more preferably not more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, and more preferably 15-20%.

[0109] More preferably, the number of the added, deleted, modified and / or substituted amino acids is 1-7, more preferably 1-5, more preferably 1-3, more preferably 1-2.

[0110] In another preferred embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0111] In another preferred embodiment, the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 9.

[0112] In a more preferred embodiment, the antibodies or recombinant proteins of the present application specifically include the VL and VH sequences, as well as the CDR and FR sequences, set forth in Table B below.

[0113] Table B Summary of Antibody Sequences of the Present Invention Encoding polynucleotides The present application also provides a polynucleotide encoding the antibody or the heavy chain variable region or the light chain variable region thereof.

[0114] Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is set forth in SEQ ID NO: 17 of the Sequence Listing; and / or, the nucleotide sequence of the nucleic acid encoding the light chain variable region is set forth in SEQ ID NO: 18 of the Sequence Listing.

[0115] More preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is set forth in SEQ ID NO: 17 of the Sequence Listing; and the nucleotide sequence of the nucleic acid encoding the light chain variable region is set forth in SEQ ID NO: 18 of the Sequence Listing.

[0116] The method for preparing the nucleic acid is a conventional method in the art, and preferably comprises the following steps: obtaining the nucleic acid molecule encoding the above-mentioned protein by gene cloning technology, or obtaining the nucleic acid molecule encoding the above-mentioned protein by artificial full-sequence synthesis method.

[0117] As known by those skilled in the art, the base sequence encoding the amino acid sequence of the above-mentioned protein can be appropriately introduced with substitutions, deletions, changes, insertions or additions to provide a homolog of a polynucleotide. The homolog of the polynucleotide in the present application can be prepared by substituting, deleting or adding one or more bases of the gene encoding the sequence of the protein within the range of maintaining the activity of the antibody.

[0118] Preparation of antibodies The DNA molecule of the antibody or fragment thereof of the present application can be obtained by conventional techniques, such as by PCR amplification or screening of genomic libraries, etc. Furthermore, the coding sequences of the light and heavy chains can be fused together to form a single chain antibody.

[0119] Once the relevant sequences have been obtained, they can be obtained in large quantities by recombinant means. This is typically done by cloning them into vectors, which are then introduced into cells, and then isolating the relevant sequences from the propagated host cells by conventional means.

[0120] Furthermore, the relevant sequences can be synthesized by artificial synthesis, especially when the fragments are relatively short. Typically, longer fragments are obtained by first synthesizing a number of smaller fragments, which are then ligated together.

[0121] At present, it is possible to obtain the DNA sequences encoding the antibodies (or fragments thereof, or derivatives thereof) of the present application entirely by chemical synthesis. The DNA sequences can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can be introduced into the protein sequences of the present application by chemical synthesis.

[0122] The present application also relates to vectors comprising the appropriate DNA sequences described above, together with appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells, so that they are able to express the proteins.

[0123] The host cells can be prokaryotic cells, such as bacterial cells, or lower eukaryotic cells, such as yeast cells, or higher eukaryotic cells, such as mammalian cells. Preferred animal cells include (but are not limited to): CHO cells, HEK-293 cells.

[0124] Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present application. The antibodies of the present application are then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, size exclusion chromatography, or affinity chromatography, as well as other conventional separation and purification methods known to those skilled in the art.

[0125] The resulting monoclonal antibodies can be identified using conventional means. For example, the binding specificity of the monoclonal antibodies can be determined using immunoprecipitation or an in vitro binding assay, such as a radioimmunoassay (RIA) or an enzyme-linked immunoabsorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, using the Scatchard analysis of Munson et al., Anal. Biochem., 107: 220 (1980).

[0126] The antibodies of the present application can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If desired, the antibodies can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic lysis, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and other various liquid chromatography techniques, and combinations of these methods.

[0127] Applications The present application also provides the use of the antibodies of the present application, for example, for specifically detecting the content of TNFα antigen in TNFα-CRM197 vaccine.

[0128] Preferably, the antibodies of the present application can form an antibody pair with adalimumab for a double antibody sandwich enzyme-linked immunosorbent assay (ELISA), so as to detect the content of TNFα antigen in TNFα-CRM197 vaccine. In the antibody pair, the antibody of the present application can serve as a coating antibody, and adalimumab serves as a detection antibody; or, in the antibody pair, adalimumab can serve as a coating antibody, and the antibody of the present application serves as a detection antibody.

[0129] Main advantages of the present application 1. The monoclonal antibody (21M2 antibody) or antigen-binding fragment thereof provided by the present application has higher affinity to TNFα-CRM197 vaccine than to TNFα.

[0130] 2. The monoclonal antibody (21M2 antibody) or antigen-binding fragment thereof provided by the present application, in combination with adalimumab, can establish a method for specifically detecting the relative content of TNFα antigen in TNFα-CRM197 vaccine by using a double antibody sandwich method. This detection method is almost not responsive to free TNFα, and thus can specifically detect TNFα antigen in TNFα-CRM197 vaccine.

[0131] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods in the following examples, for which specific conditions are not indicated, are generally carried out according to conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0132] Example 1: Screening process of candidate monoclonal antibodies The main steps of the embodiment include animal immunization, cell fusion, screening of hybridoma cells, cloning of hybridoma cells, and the like.

[0133] (1) Animal immunization: 6-week-old BALB / c mice were used, and the antigen was TNFα-CRM197 vaccine. After mixing and emulsifying 10 μg of TNFα-CRM197 vaccine with Montanide ISA51VG adjuvant (France Seppic Chemical Reagent Co., Ltd., item number: T02621) in the same volume, the mixture was injected subcutaneously on the back of the mouse; the administration was performed once a week, and a total of 4 times, and one week after the last immunization, the spleen was taken for fusion.

[0134] (2) Cell fusion: the spleen cell suspension was prepared one week after the last immunization; Preparation of spleen cell suspension: prepared on the day of cell fusion, the BALB / c mice immunized with TNFα-CRM197 vaccine were taken, the eyeball was removed to collect blood, and the serum was separated as a positive control serum for antibody detection, and the mouse was killed by cervical dislocation, and the spleen was taken to prepare the spleen cell suspension.

[0135] Preparation of myeloma cell suspension: the cells were recovered two weeks in advance to ensure that the cells were in the logarithmic growth phase when used; Preparation of feeder layer cells: mouse peritoneal macrophages were obtained two days in advance and added to the 96-well plate for culture; Cell fusion: PEG-mediated cell fusion method was used, the spleen cells and myeloma cells were mixed in a ratio of 5:1 in serum-free DMEM medium, centrifuged at 1500 rpm for 5 minutes, the supernatant was removed, the centrifuge tube was shaken vigorously, the cells were shaken, 1 ml of 50% PEG (pH 8.0, 40°C) was added within 1 minute, the shaking was continued, after the addition was completed, it was allowed to stand for 90 seconds, the fusion was terminated by adding serum-free DMEM medium, centrifuged at 1500 rpm for 5 minutes, the precipitate was suspended in HAT medium, and then distributed into 96-well cell plates containing feeder cells, and cultured in a cell incubator at 37°C and 5% CO2.

[0136] (3) Screening of hybridoma cells After 5 days of cell culture, the HAT medium was replaced once, and the HT medium was replaced on the 10th day, and when the fusion cells covered 10%-50% of the bottom of the well, Western blot method was used to screen positive wells, the hybridoma cell culture supernatant was used to detect TNFα, and the secondary antibody was enzyme-labeled goat anti-mouse antibody, and the candidate hybridoma cells with high antibody expression were screened out, and the screening results are shown in Figure 1 .

[0137] As Figure 1As shown, 6 positive clones were screened from 14 clones, and the positive clone numbers were: 21M2, 5A9, 11D1, 12B9, 18E7, 21H2, and the remaining 8 clones were negative clones.

[0138] (4) Hybridoma cell cloning Prepare the hybridoma cell suspension to be cloned, dilute to 8 cells / ml with HT medium containing 20% serum, and add mouse peritoneal cells to the hybridoma cell suspension, 5E4 cells per milliliter. Seed 0.1 ml of cell suspension per well, i.e. 0.8 hybridoma cells per well; culture at 37°C in a 5% CO2 cell incubator for 7-10 days, and detect the antibody when visible colonies appear; observe under an inverted microscope and mark the wells with only single colony growth, and take the supernatant for indirect ELISA to detect TNFα and TNFα-CRM197 vaccine, respectively, using TNFα and TNFα-CRM197 vaccine to coat the plate, hybridoma cell culture supernatant (diluted 100 times) as the primary antibody, and enzyme-labeled sheep anti-mouse antibody as the secondary antibody, as described in Figure 2 and Figure 3 , and combine the Western blot results to screen four hybridoma cell strains with high expression or high affinity of anti-TNFα and anti-TNFα-CRM197 vaccine antibodies, numbered 5A9, 11D1, 12B9, and 21M2.

[0139] Example 2: Preparation of candidate antibodies The four hybridoma cell strains screened in Example 1 were separately expanded in culture, and 6-week-old nude mice were used. The nude mice were injected with 0.5 ml of liquid paraffin in the peritoneal cavity one week in advance, and the hybridoma cells obtained in step (4) of Example 1 were adjusted to 5×10 6 / ml with normal saline, and 200 μl was injected intraperitoneally. After 7-10 days, the mouse ascites was collected. The ascites was purified by Protein A chromatography column, first equilibrated the column with A liquid PB pH 7.0, then the ascites sample was loaded onto the column, linear elution was performed with B liquid 0.1 M glycine-HCl pH 2.7 until the peak of the antibody appeared, finally the pH of the purified antibody was adjusted to 7.2 with 1M pH 8.0 Tris-HCl, and the membrane was sterilized to obtain the purified four monoclonal antibodies.

[0140] Example 3: Screening of high-affinity antibodies for TNFα-CRM197 vaccine and establishment of a double antibody sandwich assay The four antibodies 5A9, 11D1, 12B9, 21M2 are labeled as 1, 2, 3, 4 respectively, adalimumab is labeled as 5, the coating antibody in the commercial TNFα content ELISA detection kit (Sino Biological, item number: SEKA10602) is labeled as 6, and the detection antibody is labeled as 7H. The antibodies 1-5 are coupled with HRP using the following method, and the coupling products are labeled as 1H, 2H, 3H, 4H, 5H respectively: (a) Detect the protein concentration of the protein that needs to be coupled, and prepare the subsequent required reagents according to the protein concentration.

[0141] (b) Weigh 66.6 mg of HRP protein into 20 ml of water (the ratio of the number of molecules of the two proteins is 1:1). The solution is brown.

[0142] (c) Add 11.1 ml of 26 mg / ml NaIO4 reagent. Mix and place in the dark at 4°C for 45 min. The solution turns green.

[0143] (d) Dissolve 9 μl of ethylene glycol in 1 ml of water, and add 5.5 ml of ethylene glycol aqueous solution to the mixed solution. Place in the dark at room temperature for 30 min.

[0144] (e) Add 33.3 mg of antibody protein. Then transfer the mixed solution into a dialysis bag, seal it and place it in 5L of 50mM pH9.5 CBS solution for dialysis, with magnetic stirring at 4°C overnight.

[0145] (f) Add 1.2 ml of 6 mg / ml NaBH4 solution prepared in advance, and place at room temperature for 4 h.

[0146] (g) Dialyze the mixed solution in 1* PBS solution, with magnetic stirring at 4°C overnight.

[0147] (h) Pass through a 75 molecular sieve column, and determine whether coupling has occurred according to the peak graph. In theory, there is one peak in the experimental group and two peaks in the negative control.

[0148] Experimental group: 500 μl of sample.

[0149] Negative control: 2.4 mg of HRP protein + 1.2 mg of antibody protein, without operation, mix and load 500 μl.

[0150] Use antibodies 1-6 for labeling, and 1H-5H, 7H as detection antibodies for pairwise combination, respectively, to screen the detection sensitivity of TNFα-CRM197 vaccine using double antibody sandwich ELISA method, and compare the affinity of antibodies with detection ability.

[0151] The specific ELISA method for detection is as follows: Dilution Buffer: 0.1% BSA in Wash Buffer pH 7.2 - 7.4, 0.2 μm membrane filtered.

[0152] Wash Buffer: 0.05% Tween20 in PBS (PBST), pH 7.2 - 7.4.

[0153] Blocking Buffer: 2% BSA in Wash Buffer.

[0154] Dilution Buffer: 0.1% BSA in Wash Buffer pH 7.2 - 7.4, 0.2 μm membrane filtered.

[0155] Substrate Solution: purchased as a finished product.

[0156] Stop Solution: 2N H2SO4.

[0157] Experimental Methods: (a) Plate coating: Add 2 μg / ml of capture antibody to each well, incubate at 4°C overnight.

[0158] (b) Wash: Wash the plate with at least 300 μl of wash buffer, run the program 2 times for a total of 3 repeats. After washing, invert the plate to remove residual wash buffer.

[0159] (c) Block: Add 300 μl of blocking buffer to the desired wells, block at room temperature for 2 h.

[0160] (d) Wash: Wash the plate with at least 300 μl of wash buffer, run the program 2 times for a total of 3 repeats. After washing, invert the plate to remove residual wash buffer.

[0161] (e) Add sample: Add 100 μl of sample or standard to each well, incubate at room temperature for 2 h.

[0162] (f) Wash: Wash the plate with at least 300 μl of wash buffer, run the program 2 times for a total of 3 repeats. After washing, invert the plate to remove residual wash buffer.

[0163] (g) Detection: Add 0.5 μg / ml of detection antibody to each well. Incubate at room temperature for 1 h.

[0164] (h) Wash: Wash the plate with at least 300 μl wash buffer, run the program for 2 times, repeat for 3 times. After washing, invert the plate to tap the residual wash buffer inside the plate.

[0165] (i) Substrate reaction: Add 200 μl of the prepared substrate solution to each well, no need to mix, avoid light, and react at room temperature for 20 min.

[0166] (j) Reaction termination: Add 50 μL of the Stop Solution to each well to terminate the reaction.

[0167] (k) Detection: Immediately after the reaction termination, detect the absorbance value at 450 nm. Analyze the standard curve by 4-parameter method.

[0168] The results are as follows (see Table 1 and Table 2): ① The double antibody sandwich ELISA method using four self-made antibody combinations of antibody pairs has no response to TNFα and TNFα-CRM197 vaccine, indicating that the four antibodies have similar binding sites with TNFα.

[0169] ② The present application unexpectedly found that the detection sensitivity of the double antibody sandwich ELISA method using four self-made antibodies 5A9, 11D1, 12B9, and 21M2 combined with HRP-conjugated adalimumab antibody pairs for detecting TNFα-CRM197 vaccine is high, while the sensitivity for detecting TNFα is low. The possible reason for this result is that in natural TNFα, the binding sites or spatial positions of adalimumab and the four self-made antibodies may exist spatial hindrance, while in TNFα-CRM197 vaccine, the spatial conformation of the conjugated TNFα may change, resulting in the self-made antibodies and adalimumab can simultaneously bind to the TNFα in the conjugate.

[0170] ③ The double antibody sandwich ELISA method using adalimumab combined with the coating antibody and detection antibody of the commercial kit (Yiqiaoshenzhou, item number: SEKA10602) in pairs has high sensitivity for detecting TNFα and TNFα-CRM197 vaccine, but the sensitivity difference for detecting the two samples is small, so it is difficult to realize the specific detection of the TNFα antigen content in the conjugate in the TNFα-CRM197 vaccine.

[0171] (4) The four antibodies 5A9, 11D1, 12B9 and 21M2 were used as coating antibodies, and the HRP-conjugated adalimumab was used as a detection antibody to detect the sensitivity of the TNFα-CRM197 vaccine by double antibody sandwich ELISA. The double antibody sandwich ELISA detection method using the combination of the antibody 21M2 and the adalimumab had the highest detection sensitivity for the TNFα-CRM197 vaccine. Therefore, the combination of the antibody 21M2 and the adalimumab can be used for the specific detection of the content of the TNFα antigen in the conjugate in the TNFα-CRM197 vaccine.

[0172] (5) When the double antibody sandwich ELISA detection method was used to detect the content of the TNFα antigen in the TNFα-CRM197 conjugate, the combination of the antibody 21M2 and the adalimumab, whether the antibody 21M2 was used as a coating antibody and the adalimumab was used as a detection antibody (i.e., 21M2-adalimumab in Table 2) or the adalimumab was used as a coating antibody and the antibody 21M2 was used as a detection antibody (i.e., adalimumab-21M2 in Table 2), could achieve the specific detection of the content of the TNFα antigen in the TNFα-CRM197 conjugate.

[0173] Table 1 Screening results of detection antibodies for the TNFα antigen in the TNFα-CRM197 vaccine Table 2 Summary of the affinity of the combination of the antibody 21M2 and the adalimumab for TNFα and the TNFα-CRM197 conjugate Example 4: Sequencing and Identification of 21M2 Through sequencing of the hybridoma cells expressing the antibody 21M2, the results showed that: the amino acid sequence of the VL-CDR1 of the light chain of the monoclonal antibody was SEQ ID NO: 10, the amino acid sequence of the VL-CDR2 of the light chain was SEQ ID NO: 11, and the amino acid sequence of the VL-CDR3 of the light chain was SEQ ID NO: 12; the amino acid sequence of the VH-CDR1 of the heavy chain of the monoclonal antibody was SEQ ID NO: 2, the amino acid sequence of the VH-CDR2 of the heavy chain was SEQ ID NO: 3, and the amino acid sequence of the VH-CDR3 of the heavy chain was SEQ ID NO: 4.

[0174] The SEQ ID NO: 10 was as follows: RASQDISNYLN The SEQ ID NO: 11 was as follows: YTSRLHS The SEQ ID NO: 12 was as follows: QQGNTLPWT The SEQ ID NO: 2 is as follows: SYYMY The SEQ ID NO: 3 is as follows: GINPSNGGTNFNEKFKS The SEQ ID NO: 4 is as follows: EVGAMDY Example 5: Combination of monoclonal antibody 21M2 and adalimumab for in vitro potency of TNFα-CRM197 vaccine Legal Verification 1. Reproducibility Using monoclonal antibody 21M2 as coating antibody, HRP-conjugated adalimumab as detection antibody, the initial concentration of TNFα-CRM197 vaccine was 160 μg / ml, and the gradient dilution was repeated three times. The dose-response curve was used to compare the EC 50 The relative antigen amount (in vitro relative titer) between batches was calculated, and the results of three tests of the relative antigen amount of the test product were compared to evaluate the reproducibility of the method. The ELISA detection method is described in detail in Example 3.

[0175] As shown in Table 3, the dose-response curve EC 50 was 0.144, 0.174, and 0.181 μg / ml, respectively, and the relative antigen amount (relative titer) of the three repetitions was 99%, 96%, and 93%, respectively, with a CV value of 3%, meeting the requirements for method reproducibility.

[0176] Table 3 Results of three tests of the relative antigen amount of the test product TNFα-CRM197 vaccine using the double-antibody sandwich ELISA method with monoclonal antibody 21M2 and adalimumab combination 2. Accuracy evaluation Using monoclonal antibody 21M2 as coating antibody, HRP-conjugated adalimumab as detection antibody, TNFα-CRM197 vaccine diluted to 250 μg / ml was used as a reference for relative TNFα antigen amount, and then diluted to 64%, 80%, 100%, 125%, and 156% levels, respectively. The relative bias RB of the relative TNFα antigen amount determination value of each level was calculated. The ELISA detection method is described in detail in Example 3. As shown in Table 4, the deviation of the test results of the five different concentration levels of TNFα-CRM197 vaccine from the theoretical relative content was less than 10%, meeting the requirements for method accuracy.

[0177] Table 4 Accuracy of the double-antibody sandwich ELISA method using the combination of monoclonal antibody 21M2 and adalimumab in detecting the relative antigen content of TNFα-CRM197 vaccine 3. Specificity Assessment (Addition of TNFα) Using monoclonal antibody 21M2 as coating antibody and HRP-coupled adalimumab as detection antibody, TNFα-CRM197 vaccine was added with 10% and 40% TNFα, respectively. The starting concentration of each sample was 160 μg / ml, and the samples were serially diluted. The dose response curve was tested and the EC values ​​were compared. 50 Calculate the relative antigen amount (in vitro relative titer) between batches. See Example 3 for details of the ELISA detection method.

[0178] As shown in Table 5, the dose-response curves of TNFα-CRM197 vaccine and the EC values ​​of 20% and 40% TNFα added, respectively, were 50 The relative antigen amounts (relative potency) of the vaccine mixtures with 20% and 40% TNFα added were 18.5, 23.4, and 29.8 μg / ml, respectively. The relative antigen amounts (relative potency) of the vaccine mixtures with 20% and 40% TNFα added were calculated to be 79% and 62%, respectively, which were almost consistent with the relative content of TNFα in the theoretical vaccine conjugate. The addition of free TNFα had no effect on the relative antigen amount of the detected vaccine, meeting the method specificity requirements.

[0179] Table 5 Relative antigen content of TNFα-CRM197 vaccine supplemented with TNFα detected by double-antibody sandwich ELISA using monoclonal antibody 21M2 combined with adalimumab Example 6: Combination of monoclonal antibody 21M2 and adalimumab for the in vitro potency of TNFα-CRM197 vaccine Conformity Assessment Monoclonal antibody 21M2 was used as the coating antibody and HRP-conjugated adalimumab was used as the detection antibody. The starting concentration of TNFα-CRM197 vaccine was 160 μg / ml. The dose-response curves of three batches of TNFα-CRM197 vaccine (denoted as 20220501, 20220502, and 20220503) were tested by serial dilution. The EC values ​​were compared. 50 Calculate the relative antigen amount (in vitro relative titer) between batches. See Example 3 for details of the ELISA detection method.

[0180] like Figure 4 The results showed that the dose response curves of the three batches of TNFα-CRM197 vaccine EC 50The relative antigen amount (relative titer) of the three batches were 100%, 111%, 114% respectively, which had good batch consistency. Therefore, the monoclonal antibody 21M2 combined with adalimumab can be used for the consistency evaluation of the in vitro titer of TNFα-CRM197 vaccine.

[0181] All documents referred to in the present application are incorporated herein by reference as if each individual document were incorporated by reference. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that each disclosed embodiment can be implemented with or without the corresponding benefits disclosed herein.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof that recognizes the TNFα antigen in the TNFα-CRM197 vaccine, characterized in that: The heavy chain variable region (VH) of the antibody or antigen-binding fragment thereof has the following complementarity determining regions (CDRs): VH-CDR1 shown in SEQ ID NO: 2, VH-CDR2 shown in SEQ ID NO: 3, and VH-CDR3 shown in SEQ ID NO: 4; The light chain variable region (VL) of the antibody or antigen-binding fragment thereof has the following complementarity determining regions (CDRs): VL-CDR1 shown in SEQ ID NO: 10, VL-CDR2 shown in SEQ ID NO: 11, and VL-CDR3 shown in SEQ ID NO:

12.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof is SEQ ID NO:

9.

3. A recombinant protein, characterized in that The recombinant protein has: (i) the antibody or antigen-binding fragment thereof according to claim 1 or 2; and (ii) an optional tag sequence to facilitate expression and / or purification.

4. A polynucleotide, characterized in that The polynucleotide encodes the antibody or antigen-binding fragment thereof according to claim 1 or 2, or the recombinant protein according to claim 3.

5. A carrier, characterized in that The vector contains the polynucleotide according to claim 4.

6. A host cell, characterized in that The host cell contains the vector according to claim 5, or the polynucleotide according to claim 4 is integrated into its genome.

7. A detection kit, characterized in that The detection kit comprises: the antibody or antigen-binding fragment thereof according to claim 1 or 2 or the recombinant protein according to claim 3.

8. The detection kit according to claim 7, characterized in that The detection kit further comprises: adalimumab.

9. A method for preparing a recombinant polypeptide, wherein the recombinant polypeptide is the antibody or antigen-binding fragment thereof according to claim 1 or 2 or the recombinant protein according to claim 3, characterized in that: The method comprises the steps of: (a) culturing the host cell according to claim 6 under conditions suitable for expression, thereby obtaining a culture containing the recombinant polypeptide; and (b) isolating and / or purifying the recombinant polypeptide from the culture.

10. Use of the antibody or antigen-binding fragment thereof according to claim 1 or 2, the recombinant protein according to claim 3, the polynucleotide according to claim 4, the vector according to claim 5, or the host cell according to claim 6, characterized in that: Used for preparing a detection reagent or a detection kit, wherein the detection reagent or the detection kit is used for detecting the content of TNFα antigen in TNFα-CRM197 vaccine.

11. A method for detecting the content of TNFα antigen in TNFα-CRM197 vaccine, characterized in that: The detection method comprises the steps of: (S1) Provide samples to be tested; (S2) The antibody or antigen-binding fragment thereof according to claim 1 or 2, or the recombinant protein according to claim 3, is combined with adalimumab to form an antibody pair, and the sample to be tested is subjected to a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) to detect the content of TNFα antigen in the TNFα-CRM197 vaccine.

12. Use of the antibody or antigen-binding fragment thereof according to claim 1 or 2, the recombinant protein according to claim 3, or the detection kit according to claim 7 or 8, characterized in that: Assay for detecting the TNFα antigen content in the TNFα-CRM197 vaccine.

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