Anti-diphtheria toxin mutant antibody and its preparation method and application

Abstract

The present invention provides an anti-diphtheria toxin mutant antibody, its preparation method, and application. The anti-diphtheria toxin mutant antibody (anti-CRM197 antibody) obtained through screening has high affinity for free CRM197 but weak affinity for conjugated CRM197, resulting in good discrimination. Therefore, it can be used to detect the content of free CRM197 in CRM197 conjugate vaccines. Based on this, a method has been developed for quantitatively detecting the content of unconjugated CRM197 in conjugate vaccines using CRM197 as a carrier protein, thereby establishing a quality control method for the free CRM197 content in CRM197 conjugate vaccines.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to an anti-diphtheria toxin mutant antibody and a preparation method and application thereof. Background Art

[0002] CRM197 (cross-reacting materials 197) is a diphtheria toxin mutant, originally obtained from a strain with a mutated virulence gene of Corynebacterium diphtheriae. Due to a missense mutation, glycine (Gly) at position 52 in the diphtheria toxin antigen-binding fragment is mutated to glutamic acid (Glu), altering the diphtheria toxin enzyme active site. The A fragment of CRM197 is unable to bind to EF2 and, consequently, is incapable of cytotoxicity. However, its immunogenicity is not significantly different from that of diphtheria toxin. Both CRM197 and DT can bind to the diphtheria toxin receptor (DTR) on the cell membrane. Therefore, CRM197 is widely used in vaccine development as an ideal immune protein carrier.

[0003] CRM197 conjugate vaccines are typically large molecular weight, complex mixtures, with residual free CRM197 being a key quality attribute for these vaccines. However, for protein-CRM197 conjugates, identifying unconjugated CRM197 is challenging due to the similar properties of free CRM197 and CRM197 in the conjugate.

[0004] Therefore, developing an antibody that can specifically recognize free CRM197 in CRM197 conjugate vaccines is an urgent need in the field to solve the key problem of quality control of CRM197 conjugate vaccines. Summary of the Invention

[0005] The purpose of the present invention is to provide an anti-diphtheria toxin mutant (CRM197) antibody, which can bind to CRM197 with high affinity but lacks affinity for CRM197 conjugate vaccine and can be used to detect the content of free CRM197 in CRM197 conjugate vaccine.

[0006] In the first aspect of the present invention, an anti-diphtheria toxin mutant CRM197 antibody or an antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof has the 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 the complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3 contained in the light chain variable region (VL) shown in SEQ ID NO: 9.

[0007] In another preferred embodiment, the complementarity determining regions (CDRs) are defined according to the IMGT, Kabat or Chothia numbering systems.

[0008] In another preferred embodiment, the heavy chain variable region (VH) of the antibody or antigen-binding fragment thereof has the following complementarity determining region CDR:

[0009] 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(NY);

[0010] The light chain variable region of the antibody or antigen-binding fragment thereof has the following complementarity determining regions CDR:

[0011] 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;

[0012] Among them, any of the above amino acid sequences also includes a derivative sequence obtained by optionally adding, deleting, modifying and / or replacing at least one (such as 1-3) amino acid.

[0013] In another preferred embodiment, the sequence identity of the derived sequence to any of the above amino acid sequences is 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%.

[0014] 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.

[0015] In another preferred example, the amino acid sequence of the VH-FR1 is shown in SEQ ID NO: 5, the amino acid sequence of the VH-FR2 is shown in SEQ ID NO: 6, the amino acid sequence of the VH-FR3 is shown in SEQ ID NO: 7, and the amino acid sequence of the VH-FR4 is shown in SEQ ID NO: 8.

[0016] 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.

[0017] In another preferred example, the amino acid sequence of the VL-FR1 is shown in SEQ ID NO: 13, the amino acid sequence of the VL-FR2 is shown in SEQ ID NO: 14, the amino acid sequence of the VL-FR3 is shown in SEQ ID NO: 15, and the amino acid sequence of the VL-FR4 is shown in SEQ ID NO: 16.

[0018] 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.

[0019] In another preferred example, the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof is SEQ ID NO: 9.

[0020] In another preferred embodiment, the antibody or antigen-binding fragment thereof comprises the VL and VH sequences shown in Table B.

[0021] In another preferred embodiment, the antibody or antigen-binding fragment thereof comprises the CDR and FR sequences shown in Table B.

[0022] In another preferred embodiment, the antibody or antigen-binding fragment thereof includes a monomer, a bivalent antibody, and / or a multivalent antibody.

[0023] In another preferred embodiment, the bivalent antibody is a bispecific antibody.

[0024] In another preferred embodiment, the multivalent antibody is a multispecific antibody.

[0025] In another preferred embodiment, the antibody or antigen-binding fragment thereof includes a murine antibody, a humanized antibody or a chimeric antibody.

[0026] In another preferred embodiment, the antibody or antigen-binding fragment thereof is a partially or fully humanized, or fully human monoclonal antibody.

[0027] In another preferred embodiment, the antigen-binding fragment is selected from scFv, Fab, Fab', F(ab')2, Fv fragment, heavy chain antibody, and disulfide-linked Fv (dsFv).

[0028] In another preferred embodiment, the heavy chain constant region of the antibody or antigen-binding fragment thereof is selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4.

[0029] In another preferred embodiment, the light chain constant region of the antibody or antigen-binding fragment thereof is selected from the constant region of a human antibody κ chain or λ chain.

[0030] In a second aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein has:

[0031] (i) the antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and

[0032] (ii) an optional tag sequence to facilitate expression and / or purification.

[0033] In another preferred example, the tag sequence includes an Fc tag, an HA tag, a GGGS sequence, a FLAG tag, a Myc tag, a 6His tag, or a combination thereof.

[0034] In another preferred embodiment, the recombinant protein includes a fusion protein.

[0035] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a multimer.

[0036] In a third aspect of the present invention, a polynucleotide is provided, which encodes the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the recombinant protein according to the second aspect of the present invention.

[0037] In another preferred embodiment, the polynucleotide is RNA (including mRNA) or DNA (including cDNA).

[0038] In another preferred example, the polynucleotide comprises: a heavy chain variable region encoding sequence as shown in SEQ ID NO: 17, and a light chain variable region encoding sequence as shown in SEQ ID NO: 18.

[0039] In the fourth aspect of the present invention, a vector is provided, wherein the vector contains the polynucleotide according to the third aspect of the present invention.

[0040] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

[0041] In another preferred embodiment, the vector comprises a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

[0042] In another preferred embodiment, the vector is selected from the following group: pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV, etc.

[0043] In another preferred embodiment, the vector further comprises elements selected from the following group: a promoter, a transcription enhancing element WPRE, a long terminal repeat sequence LTR, etc.

[0044] In the fifth aspect of the present invention, a host cell is provided, wherein the host cell contains the vector according to the fourth aspect of the present invention, or the polynucleotide according to the third aspect of the present invention is integrated into its genome.

[0045] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0046] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0047] In a sixth aspect of the present invention, a detection kit is provided, comprising: the antibody or antigen-binding fragment thereof according to the first aspect of the present invention or the recombinant protein according to the second aspect of the present invention as a coating antibody.

[0048] In another preferred embodiment, the detection kit further comprises: enzyme-labeled diphtheria toxin (DT) antibody as a detection antibody.

[0049] In another preferred embodiment, the enzyme-labeled diphtheria toxin (DT) antibody is: a diphtheria toxin (DT) antibody coupled with horseradish peroxidase (HRP), or a diphtheria toxin (DT) antibody coupled with alkaline phosphatase (ALP).

[0050] In another preferred embodiment, the detection kit is a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit.

[0051] In another preferred embodiment, the detection kit further comprises: a CRM197 protein standard.

[0052] In another preferred example, the amino acid sequence of the CRM197 protein is shown in SEQ ID NO: 19.

[0053] In another preferred embodiment, the CRM197 protein standard is a serially diluted standard, and the concentrations of the standard include: 1000 μg / ml, 500 μg / ml, 250 μg / ml, 125 μg / ml, 62.5 μg / ml, 31.25 μg / ml and 15.625 μg / ml.

[0054] In another preferred embodiment, the detection kit further includes: a color developing solution (i.e., an enzyme substrate solution, such as TMB commonly used for HRP and PNPP commonly used for ALP), a stop solution (such as H2SO4 commonly used for HRP reaction and NaOH solution commonly used for ALP reaction), a sample diluent, a positive control, a negative control, or a combination thereof.

[0055] In a seventh aspect of the present invention, a method for preparing a recombinant polypeptide is provided, wherein the recombinant polypeptide is the antibody or antigen-binding fragment thereof according to the first aspect of the present invention or the recombinant protein according to the second aspect of the present invention, the method comprising the steps of:

[0056] (a) culturing the host cell according to the fifth aspect of the present invention under conditions suitable for expression, thereby obtaining a culture containing the recombinant polypeptide; and

[0057] (b) isolating and / or purifying the recombinant polypeptide from the culture.

[0058] In the eighth aspect of the present invention, there is provided a 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, the polynucleotide as described in the third aspect of the present invention, the vector as described in the fourth aspect of the present invention, or the host cell as described in the fifth aspect of the present invention for preparing a detection reagent or a detection kit.

[0059] In another preferred embodiment, the detection kit is the detection kit as described in the sixth aspect of the present invention.

[0060] In another preferred embodiment, the detection reagent or detection kit is used to detect the content of unconjugated free CRM197 in a conjugate vaccine with CRM197 as a carrier.

[0061] In another preferred embodiment, the conjugate vaccine includes: TNFα-CRM197 vaccine, recombinant hEGF-CRM197 tumor therapeutic vaccine, a conjugate vaccine obtained by cross-linking other proteins with CRM197, or a combination thereof.

[0062] In a ninth aspect of the present invention, a method for detecting the content of free CRM197 that has not been conjugated in a conjugate vaccine using CRM197 as a carrier is provided, the method comprising the steps of:

[0063] (S1) Provide samples to be tested;

[0064] (S2) Using the antibody or antigen-binding fragment thereof described in the first aspect of the present invention or the recombinant protein described in the second aspect of the present invention as a coating antibody and an enzyme-labeled diphtheria toxin (DT) antibody as a detection antibody, the sample to be tested is subjected to a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) to detect the content of free CRM197.

[0065] In another preferred embodiment, the detection method further comprises the step of providing a detection kit as described in the sixth aspect of the present invention.

[0066] In another preferred embodiment, the sample to be tested includes: one or more conjugate vaccines using CRM197 as a carrier.

[0067] In another preferred embodiment, the sample to be tested includes: TNFα-CRM197 vaccine, recombinant hEGF-CRM197 tumor therapeutic vaccine, a combination vaccine of other proteins and CRM197 cross-linked, or a combination thereof.

[0068] In another preferred embodiment, the enzyme-labeled diphtheria toxin (DT) antibody is: a diphtheria toxin (DT) antibody coupled with horseradish peroxidase (HRP), or a diphtheria toxin (DT) antibody coupled with alkaline phosphatase (ALP).

[0069] In another preferred embodiment, the detection method comprises the steps of:

[0070] Step 1: coating the antibody or antigen-binding fragment thereof according to the first aspect of the present invention or the recombinant protein according to the second aspect of the present invention on an ELISA plate at a certain concentration;

[0071] Step 2: Block the ELISA plate with blocking solution;

[0072] Step 3: Dilute the sample to be tested and the CRM197 protein standard in series and incubate them on the ELISA plate;

[0073] Step 4: Block the ELISA plate with blocking solution;

[0074] Step 5: Add HRP-labeled diphtheria toxin antibody to each reaction well and incubate at 37°C for 1 hour;

[0075] Step 6: Add color developing solution to develop color, then add stop solution to terminate the reaction; measure the absorbance at 450 nm;

[0076] Step 7: Calculate the content of free CRM197 in the sample to be tested based on the measured absorbance and the standard curve equation.

[0077] In another preferred example, the amino acid sequence of the CRM197 protein is shown in SEQ ID NO: 19.

[0078] In another preferred embodiment, the CRM197 protein standard is a serially diluted standard, and the concentrations of the standard include: 1000 μg / ml, 500 μg / ml, 250 μg / ml, 125 μg / ml, 62.5 μg / ml, 31.25 μg / ml and 15.625 μg / ml.

[0079] In another preferred embodiment, the color developing solution is TMB.

[0080] In another preferred embodiment, the stop solution is H2SO4.

[0081] In another preferred embodiment, the detection method further comprises the steps of fitting a four-parameter logistic curve using the CRM197 standard protein concentration (μg / ml) as the abscissa and the OD450 average value as the ordinate to obtain a standard curve equation.

[0082] In the tenth aspect of the present invention, a method for detecting the content of unconjugated free CRM197 in a conjugate vaccine with CRM197 as a carrier 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.

[0083] In another preferred embodiment, the detection method is the detection method described in the ninth aspect of the present invention.

[0084] 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

[0085] Figure 1 The results of ELISA screening of hybridoma cell lines are shown.

[0086] Figure 2 The dose-response curves of CRM197 detection using a double antibody sandwich assay with three candidate CRM197 antibodies and a diphtheria toxin antibody are shown, wherein the abscissa represents the CRM197 concentration (ng / ml) and the ordinate represents the OD450 value.

[0087] Figure 3 The dose-response curves of TNFα-CRM197 vaccine using the double antibody sandwich method with three candidate CRM197 antibodies and diphtheria toxin antibody are shown, wherein the abscissa is the CRM197 concentration (ng / ml) and the ordinate is the OD450 value.

[0088] Figure 4 A comparison chart showing the double antibody sandwich method for detecting CRM197 and TNFα-CRM197 vaccines using three candidate CRM197 antibodies and diphtheria toxin antibody, respectively.

[0089] Figure 5 The four-parameter logistic curve and equation fitted in Example 4 are shown. DETAILED DESCRIPTION

[0090] After extensive and in-depth research and extensive screening, the inventors have developed an antibody against a diphtheria toxin mutant (CRM197), provided a method for preparing the antibody and an antibody sequence, and further screened for antibodies with high affinity for CRM197 and weak affinity for the CRM197 conjugate vaccine. The antibody of the present invention has the complementary 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 the complementary 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 invention includes the VL and VH sequences shown in Table B, as well as the CDR and FR sequences. Based on the antibody of the present invention as a coating antibody, a double antibody sandwich ELISA method for detecting free CRM197 in the CRM197 conjugate vaccine was established using a commercially available diphtheria toxin antibody as a detection antibody. The detection method established by the present invention is reliable, as shown in the verification results in Table 2, and has high accuracy and precision, which can meet the detection requirements of quantitative analysis methods for biological samples. On this basis, the present invention was completed.

[0091] the term

[0092] In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings generally understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can change. It should also be understood that the terms used herein are intended only to describe specific embodiments and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0093] Unless otherwise defined, 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 invention belongs. As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. 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.).

[0094] The three-letter and one-letter codes for amino acids used in the present invention are as described in J. biol. chem, 243, p3558 (1968).

[0095] As used herein, the term "optionally" or "optionally" means that the event or situation described subsequently may occur but need not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a specific sequence may have but need not have, and may have 1, 2, or 3.

[0096] As used herein, "sequence identity" refers to the degree of identity between two nucleic acid or amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions, or deletions. The sequence identity between a sequence described herein and a sequence to which it is identical may 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%, and 100%.

[0097] As used herein, the terms "diphtheria toxin mutant", "CRM197 protein", and "CRM197" are used interchangeably and all refer to the CRM197 protein to which the antibodies of the present invention specifically bind.

[0098] Preferably, the amino acid sequence of the diphtheria toxin mutant CRM197 of the present invention is shown in SEQ ID NO: 19:

[0099] GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASR VVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVT GTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTV EDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS (SEQ ID NO: 19)

[0100] Antibody

[0101] As used herein, the terms "antibody" or "immunoglobulin" are heterotetrameric glycoproteins of approximately 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0102] As used herein, the term "variable" refers to certain portions of the variable region of an antibody that differ in sequence, which contribute to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of native heavy and light chains each contain four FR regions, which generally have a β-pleated structure and are connected by three CDRs that form a connecting loop, which in some cases can form a partial β-pleated structure. The CDRs in each chain are closely together through the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume 1, pages 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0103] The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two distinct classes, called kappa and lambda, based on the amino acid sequences of their constant regions. Immunoglobulins can also be divided into different classes based on the amino acid sequences of their heavy chain constant regions. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to the different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different immunoglobulin classes are well known in the art.

[0104] Generally, an antibody's antigen-binding properties are described by three specific regions located in the variable regions of the heavy and light chains, known as the variable regions (CDRs). These regions are divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, spatially close to each other through the β-sheet formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antibody's antigen-binding site. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0105] In the present invention, "VH-CDR1" and "CDR-H1" are used interchangeably to refer to the CDR1 of the heavy chain variable region; "VH-CDR2" and "CDR-H2" are used interchangeably to refer to the CDR2 of the heavy chain variable region; "VH-CDR3" and "CDR-H3" are used interchangeably to refer to the CDR3 of the heavy chain variable region. "VL-CDR1" and "CDR-L1" are used interchangeably to refer to the CDR1 of the light chain variable region; "VL-CDR2" and "CDR-L2" are used interchangeably to refer to the CDR2 of the light chain variable region; "VL-CDR3" and "CDR-L3" are used interchangeably to refer to the CDR3 of the light chain variable region.

[0106] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0107] The term "epitope" or "antigenic determinant" refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation.

[0108] The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. -7 M, for example, less than approximately 10 -8 M, 10 -9 M or 10 -10 Binds with an affinity (KD) of M or less.

[0109] As used herein, the term "antigenic determinant" refers to a discrete three-dimensional site on an antigen that is recognized by the antibodies or antigen-binding fragments of the present invention.

[0110] In the present invention, the antibody can specifically recognize the CRM197 protein, and its affinity is significantly higher than that of commercially available antibodies.

[0111] For purposes of the present invention, antibodies include murine, chimeric, humanized, or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising human and non-human portions, can be obtained using standard recombinant DNA techniques and are all useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as a chimeric antibody having variable regions from a murine monoclonal antibody and constant regions from a human immunoglobulin (see, e.g., U.S. Patents 4,816,567 and 4,816,397, which are incorporated herein by reference in their entireties). A humanized antibody refers to an antibody molecule derived from a non-human species that has 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 recombinant DNA techniques well known in the art.

[0112] In the present invention, the antibodies can be monospecific, bispecific, trispecific, or more multispecific.

[0113] In the present invention, the antibodies of the present invention also include conservative variants thereof, which refer to polypeptides in which no more than 10, preferably no more than 8, more preferably no more than 5, and most preferably no more than 3 amino acids are replaced with amino acids having similar or similar properties, compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by making amino acid substitutions according to Table A.

[0114] Table A

[0115]

[0116] In the present invention, the antibody is an antibody that specifically binds to CRM197. The present invention provides a highly specific and high-affinity antibody against CRM197, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region (VH) amino acid sequence, and the light chain comprises a light chain variable region (VL) amino acid sequence.

[0117] The heavy chain variable region (VH) has a complementarity determining region (CDR) selected from the group consisting of:

[0118] 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 (NY);

[0119] The light chain variable region (VL) has a complementarity determining region (CDR) selected from the group consisting of:

[0120] 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 also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one amino acid, and can retain high binding affinity to free CRM197 and low binding affinity to CRM197 in a CRM197-coupled vaccine.

[0121] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or replacing at least one amino acid is preferably an amino acid sequence with a homology or sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

[0122] Methods for determining sequence homology or identity that are well known to those of ordinary skill in the art include, but are not limited to, Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). Preferred methods for determining identity are to obtain the largest possible match between the sequences tested. Methods for 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 program package (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.

[0123] Preferably, the antibodies described herein are one or more of a full-length antibody 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 monoclonal or polyclonal antibodies prepared from the above antibodies. The monoclonal antibodies can be prepared by a variety of approaches and technologies, including hybridoma technology, phage display technology, and single lymphocyte gene cloning technology. The mainstream method is to prepare monoclonal antibodies from wild-type or transgenic mice using hybridoma technology.

[0124] The full-length antibody protein is a conventional full-length antibody protein in the art, comprising 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 light chain variable region of the protein, together with the human heavy chain constant region and the human light chain constant region, constitute a fully human full-length antibody protein. Preferably, the full-length antibody protein is IgG1, IgG2, IgG3, or IgG4; more preferably, IgG1.

[0125] The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, and humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and even more preferably fully humanized antibodies.

[0126] The antibody derivatives of the present invention can be single-chain antibodies and / or antibody fragments, such as Fab, Fab', F(ab')2 or other antibody derivatives known in the art, as well as any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0127] The single-chain antibody 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 to 20 amino acids.

[0128] The animal is preferably a mammal, such as a mouse.

[0129] The antibody of the present invention may be a chimeric antibody, a humanized antibody, a CDR-grafted and / or modified antibody targeting CRM197.

[0130] In the above content of the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the original amino acid sequence, more preferably no more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, more preferably 15-20%.

[0131] In the above content of the present invention, more preferably, the number of amino acids added, deleted, modified and / or substituted may be 1-7, more preferably 1-5, more preferably 1-3, more preferably 1-2.

[0132] In another preferred embodiment, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 1.

[0133] In another preferred embodiment, the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 9.

[0134] In a more preferred embodiment, each antibody or recombinant protein of the present invention specifically includes each VL and VH sequence, as well as CDR and FR sequences shown in Table B below.

[0135] Table B Summary of antibody sequences of the present invention

[0136]

[0137] Encoding polynucleotide

[0138] The present invention also provides a polynucleotide encoding the above-mentioned antibody or its heavy chain variable region or light chain variable region.

[0139] Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID NO: 17 in the sequence listing; and / or, the nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in SEQ ID NO: 18 in the sequence listing.

[0140] More preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID NO: 17; and the nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in SEQ ID NO: 18.

[0141] The method for preparing the nucleic acid is a conventional method in the art, and preferably comprises the following steps: obtaining a nucleic acid molecule encoding the above protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above protein by artificial full sequence synthesis.

[0142] Those skilled in the art will appreciate that the base sequence encoding the amino acid sequence of the aforementioned protein can be appropriately substituted, deleted, altered, inserted, or added to provide a polynucleotide homolog. The polynucleotide homologs of the present invention can be prepared by substituting, deleting, or adding one or more bases in the gene encoding the protein sequence while maintaining antibody activity.

[0143] Antibody preparation

[0144] The sequence of the DNA molecule of the antibody of the present invention or its fragment can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences of the light chain and heavy chain can be fused together to form a single-chain antibody.

[0145] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0146] In addition, the sequences can also be synthesized by artificial synthesis, especially when the fragment length is shorter. Usually, a long fragment can be obtained by synthesizing multiple small fragments and then connecting them.

[0147] Currently, DNA sequences encoding the antibodies (or fragments or derivatives thereof) of the present invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the present invention through chemical synthesis.

[0148] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0149] Host cells can be prokaryotic cells, such as bacterial cells, 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 and HEK-293 cells.

[0150] Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present invention. The antibodies of the present invention are then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, among other conventional separation and purification methods well known to those skilled in the art.

[0151] 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 in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent 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).

[0152] The antibodies of the present invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the antibodies can be isolated and purified by various separation methods utilizing 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 shock, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques, and combinations of these methods.

[0153] application

[0154] The present invention also provides uses of the antibodies of the present invention, for example, they can be used to specifically detect the content of free CRM197 in conjugate vaccines.

[0155] Preferably, the conjugate vaccine is a TNFα-CRM197 vaccine; and / or a recombinant hEGF-CRM197 tumor therapeutic vaccine; and / or other conjugate vaccines using CRM197 as a cross-linking reactant.

[0156] Main advantages of the present invention

[0157] 1. The anti-CRM197 antibodies of the present invention possess specificity for the CRM197 epitope recognition. Unlike commercially available diphtheria toxin antibodies and other polyclonal antibodies, these antibodies possess high affinity for free CRM197 but weak affinity for CRM197-conjugated vaccines. Therefore, these antibodies can be used to detect the free CRM197 content in CRM197-conjugated vaccines.

[0158] 2. A method for detecting free CRM197 in CRM197 conjugate vaccines was established based on the anti-CRM197 antibodies of the present invention. This method addresses the quality control or research of free CRM197, a key impurity in CRM197 conjugate vaccines, and addresses a key issue in the drugability of CRM197 conjugate vaccines.

[0159] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and parts by weight.

[0160] Example 1: Preparation and screening of anti-CRM197 antibody monoclonal cell lines

[0161] BALB / c mice were immunized multiple times with CRM197 protein (the preparation method is described in the inventor's patent application: Preparation Method of Diphtheria Toxin Mutant CRM197, Patent Grant No.: CN104140972B; its amino acid sequence is (SEQ ID NO: 19)). This protein was then fused with SP2 / 0 myeloma cells to generate hybridomas. Three monoclonal cell lines were identified using ELISA and monoclonal cloning. The affinity of the antibodies in the cell supernatants for CRM197 was determined by ELISA.

[0162] (1) Materials and instruments

[0163] 1. Mice: BALB / c

[0164] 2. Myeloma cells: SP2 / 0 (ATCC)

[0165] 3. Immunogen: CRM197 protein (1 mg / ml)

[0166] 4. Cell growth medium: 1640 + 10% FBS (Gibco) + OPI Media Supplement (Sigma)

[0167] 5. Detection Antibody: M-HRP-anti-mouse IgG

[0168] 6. Cell fusion instrument: BTX ECM 2001 multifunctional cell fusion instrument

[0169] (2) Animal immunization methods

[0170] 1. Mouse lymph node immunization: CRM197 protein was injected into the inguinal lymph nodes at a dose of 50 μg per mouse, once every two weeks for a total of three immunizations. Blood was collected from the tail vein for the final immunization and titer was determined. Hybridomas were prepared when the titer was above 1:100,000.

[0171] 2. Hybridoma cell preparation: Hybridoma cells were prepared using the ECM2001 multifunctional cell fusion instrument.

[0172] (III) ELISA detection method (indirect method)

[0173] 1. CRM197 protein was diluted to 1 μg / ml in PBS, and 100 μl / well was coated at 4°C overnight.

[0174] 2. Wash the plate once with PBST and block with 200 μl / well of 3% BSA-PBS at 37°C for 1 hour.

[0175] 3. Wash the plate once with PBST;

[0176] 4. Dilute the hybridoma culture supernatant 100-fold with 1% BSA-PBS, add 100 μl / well, and incubate at 37°C for 1 hour.

[0177] 5. Wash the plate three times with PBST;

[0178] 6. Add 100 μl / well of anti-mouse IgG secondary antibody diluted in 1% BSA-PBS and incubate at 37°C for 1 hour.

[0179] 7. Wash the plate 5 times with PBST;

[0180] 8. Develop color with TMB and read the OD450 value on a microplate reader.

[0181] (IV) Results

[0182] Three hybridoma cell lines with better growth status and higher antibody detection values ​​were screened, namely 10G2, 15C2, and 16M3. The ELISA test results are shown in Figure 1 .

[0183] Example 2: Preparation of three candidate CRM197 antibodies

[0184] Experimental Methods: The three hybridoma cell lines described in Example 1 were monocloned using the limiting dilution method to generate anti-CRM197 monoclonal antibody cells. These cells were cultured and expanded, then intraperitoneally inoculated into Balb / c mice. The mice were observed for the development of ascites within 7-14 days after injection, and the ascites fluid was collected.

[0185] The collected ascites was first precipitated with ammonium sulfate, and the precipitate was resuspended in buffer A (20 mM PBS, pH 7.0) and loaded onto a Protein A antibody purification column equilibrated with buffer A. After loading, the column was rinsed with buffer A until the A280 value and conductivity returned to baseline. Elution was then performed in a single step with 100% buffer B (20 mM citric acid, pH 2.7). The A280 value of the eluate was monitored, and the eluted fractions were collected as the primary purified anti-CRM197 antibody product.

[0186] The pH of the preliminary purified product was adjusted to 5.0-6.0 using buffer C (10 mM citric acid, pH 5.5), loaded onto an already equilibrated cation exchange chromatography column, and then the NaCl concentration was linearly increased until 40% elution was achieved. The largest eluted fraction was collected, which was the anti-CRM197 antibody.

[0187] Experimental results: Ammonium sulfate treatment can quickly and effectively remove impurities from ascites. The treated crude extract is passed through a Protein A antibody column to capture anti-CRM197 antibodies in a single step. Further purification using a cation exchange column yields highly purified anti-CRM197 antibodies.

[0188] Example 3: Affinity Identification of Candidate CRM197 Antibodies to CRM197 Protein and TNF-CRM197 Vaccine

[0189] The present invention intends to screen CRM197 antibodies with significantly different affinities for CRM197 protein and TNF-CRM197 vaccine, and apply them to the detection of free CRM197 in TNF-CRM197 vaccine. Therefore, the present invention selects three candidate antibodies as coating antibodies, and a commercial HRP-conjugated diphtheria toxin (DT) antibody as the detection antibody. At the same time, a commercial CRM197 detection kit (R&D Systems, catalog number: 940-DTX-AG1) is selected to test the detection sensitivity of CRM197 protein and TNF-CRM197 vaccine, respectively.

[0190] Double antibody sandwich enzyme-linked immunosorbent assay (ELISA) is based on the specific binding characteristics of antigen and antibody and is commonly used to detect antigen / antibody content. The coated antibody is diluted in a certain ratio and coated onto a solid phase carrier. Then, the analyte of different dilution concentrations is added, followed by the diluted specific enzyme-labeled antibody. After incubation, the substrate is added to develop color and the results are interpreted.

[0191] Experimental methods:

[0192] 1. Dilute the candidate CRM197 antibody (or coating antibody) to 2 μg / ml in coating buffer. Coat the ELISA plate at 100 μl / well. Seal the plate and incubate at 4°C overnight. Discard the liquid in the ELISA plate wells. Wash the plate three times with washing buffer.

[0193] 2. TNFα-CRM197 vaccine and CRM197 were serially diluted and incubated in an ELISA plate at 100 μl / well. Seal the plate and incubate at 37°C ± 1°C for 1 hour. Discard the liquid in the plate wells and wash the plate three times with washing solution.

[0194] 3. Add 100 μl of HRP-conjugated diphtheria toxin antibody (1:1000) to each well. Vortex and mix at 37°C for 10 minutes. Seal the plate and incubate at 37°C ± 1°C for 1 hour. Wash the plate five times.

[0195] 4. Add 100 μl / well of the color development solution to the ELISA plate. After color development for 15 minutes at room temperature in the dark, add 50 μl / well of the stop solution to terminate the reaction. Measure the absorbance at 450 nm (OD450) using a microplate reader.

[0196] Experimental results:

[0197] Comparison of dose-response curves of different antibodies in sandwich ELISA:

[0198] like Figure 2 As shown, when the candidate antibody numbered 10G2 is a coating antibody (corresponding to Figure 2 Plot 2 in Figure 2) has the highest sensitivity for detecting CRM197, followed by 16M3 as the coating antibody (corresponding to Figure 2 Plot4 in Figure 4), while the commercial CRM197 detection antibody (corresponding to Figure 2 Plot1 in ) and 15C2 (corresponding to Figure 2 Plot3 in the figure shows low sensitivity.

[0199] like Figure 3 As shown, when only 10G2 is the coating antibody (corresponding to Figure 3Plot2 in Figure 3 has a high detection sensitivity for TNF-CRM197 vaccine, while the other two antibodies and commercial CRM197 detection reagents have almost no response to TNF-CRM197 vaccine.

[0200] The dose-response curves of the four antibody combinations for testing CRM197 and TNF-CRM197 vaccines are shown in the figure. Figure 4 The results showed that although the commercial detection kit or 10G2 and 15C2 had a higher affinity for CRM197 than the TNF-CRM197 vaccine when used as coating antibodies, the difference was not large enough to completely distinguish CRM197 from the TNF-CRM197 vaccine. When only 16M3 was used as the coating antibody, it had almost no response to the TNF-CRM197 vaccine within the concentration range of the CRM197 dose-response curve. Therefore, this condition can be used to detect the content of free CRM197 in the TNF-CRM197 vaccine process product.

[0201] Example 4: Establishment of free CRM197 content detection in TNF-CRM197 vaccine using the CRM197 antibody of the present invention method

[0202] The free CRM197 content in the TNF-CRM197 vaccine was detected by a double antibody sandwich method using the CMR197 antibody of the present invention and a commercial diphtheria toxin antibody. The specific operation is as follows:

[0203] 1. Add CRM197 antibody to coating solution (Na2CO3-NaHCO3 buffer) and mix to a final concentration of 12.0 μg / ml. Coat the ELISA plate at 100 μl / well. Seal the plate and incubate at 4°C overnight. Discard the coating solution and wash the plate three times with washing solution.

[0204] 2. Add 200 μl / well of blocking solution (PBS containing 0.05% (v / v) Tween-20, 1% (w / v) BSA) to the ELISA plate. Seal the plate and incubate at 37°C ± 1°C for 2 hours. Discard the blocking solution and wash the plate three times with washing solution.

[0205] 3. Add serially diluted standards (1000 μg / ml, 500 μg / ml, 250 μg / ml, 125 μg / ml, 62.5 μg / ml, 31.25 μg / ml, and 15.625 μg / ml) and the test sample to the ELISA plate at 100 μl / well in duplicate. Incubate at 37°C ± 1°C for 1 hour. Discard the liquid in the plate and wash the plate three times with washing solution.

[0206] 4. Add 100 μl of HRP-labeled mouse anti-diphtheria toxin (DT) monoclonal antibody (1:500) to each well and incubate at 37°C ± 1°C for 1 hour. Discard the liquid in the plate and wash the plate 5 times with washing solution.

[0207] 5. Add 100 μl / well of the color development solution to the microplate. Allow the color to develop at room temperature in the dark for 15–30 minutes. Then, add 50 μl / well of the stop solution to terminate the reaction. Measure the absorbance at 450 nm (OD450) using a microplate reader. Use the microplate reader's built-in four-parameter regression method to calculate the standard curve equation, using the CRM197 standard protein concentration (μg / ml) as the abscissa and the average OD450 value as the ordinate.

[0208] Experimental results:

[0209] like Figure 5 As shown in the figure, the method uses the CRM197 standard protein concentration (μg / ml) as the horizontal axis and the OD450 average value as the vertical axis between 15 μg / ml and 1000 μg / ml. The determination coefficient R2 of the fitted four-parameter logistic curve is 0.996, which has a high degree of fit.

[0210] Table 1 OD values ​​of a series of standard solutions

[0211]

[0212] The accuracy and precision of the test method were validated according to the "Guidelines for Validation of Quantitative Analysis Methods for Biological Samples" in Part IV, Section 9012 of the 2020 edition of the Chinese Pharmacopoeia. Accuracy describes the closeness of the measured value to the labeled concentration of the analyte. Accuracy should be assessed using samples spiked with a known amount of analyte, i.e., quality control samples. The average concentration of each quality control sample should be within ±20% of the labeled value (±25% for the lower and upper limits of quantitation). Precision describes the closeness of repeated measurements of the analyte. Results from the same analytical batch used to demonstrate accuracy should be used to obtain precision at the upper and lower limits of quantitation, as well as for low, medium, and high concentration quality control samples. Precision should not exceed 20% (25% for the lower and upper limits of quantitation).

[0213] The verification results are shown in Table 2. It was verified that the upper and lower limits of quantification and the recoveries of low, medium and high concentration quality control samples were all within the range of 80% to 120% (and reached a more accurate range of approximately 90% to 110%), and the coefficient of variation CV% was less than 20% (and reached a more precise range of less than 6%, or even ≤4%), indicating that the method for detecting the free CRM197 content in TNF-CRM197 vaccine using the CRM197 antibody of the present invention is reliable, can meet the detection requirements, and the detection method has high accuracy and precision.

[0214] Table 2 Summary of the accuracy and precision verification results of the detection methods

[0215]

[0216] Example 5: The method for detecting free CRM197 in the CRM197 conjugate vaccine established by the present invention was used to detect the free CRM197 content in three batches of TNF-CRM197 vaccine quality control

[0217] The quality control of three batches of TNF-CRM197 vaccine was carried out using the method for detecting the free CRM197 content in the CRM197 conjugate vaccine established in the present invention. The test results are shown in the following table. The results show that the free CRM197 content of the TNF-CRM197 vaccine is low and has good batch-to-batch consistency.

[0218] Table 3 Results of free CRM197 content detection of three batches of TNF-CRM197 vaccine

[0219]

[0220] Example 6: Detection of free CRM197 content after adding CRM197 to TNF-CRM197 vaccine and calculation of detection addition value Relative recovery rate to theoretical value

[0221] In order to further evaluate the accuracy of the double antibody sandwich method composed of CMR197 antibody and commercial diphtheria toxin antibody established in the present invention to detect the free CRM197 content in TNF-CRM197 vaccine, three known concentrations of CRM197 were added to the TC20220307 batch of TNF-CRM197 vaccine, and the free CRM197 content of the CRM197-added samples was detected according to the operating method of Example 4.

[0222] The results are shown in Table 4. When TNF-CRM197 vaccine with known CRM197 concentration was added, the recovery rate of the added free CRM197 value relative to the theoretical value was close to 100%, and the detection error was within 5%.

[0223] Table 4 Accuracy results of the detection method verified by adding tests

[0224]

[0225] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An anti-diphtheria toxin mutant CRM197 antibody or an antigen-binding fragment thereof, 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 of the antibody or antigen-binding fragment thereof has the following complementarity determining regions CDR: 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 as a coating antibody.

8. 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.

9. 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 uncoupled free CRM197 in a TNF-CRM197 coupled vaccine.

10. A method for detecting the content of unconjugated free CRM197 in a TNF-CRM197 conjugate 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 used as the coating antibody, and the enzyme-labeled diphtheria toxin (DT) antibody is used as the detection antibody. The sample to be tested is subjected to a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) to detect the content of free CRM197.

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