CN108610417B - Anti-tetanus toxin neutralizing antibody, preparation method and application thereof - Google Patents
Anti-tetanus toxin neutralizing antibody, preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an anti-tetanus toxin neutralizing antibody, a preparation method and application thereof, wherein the amino acid sequence of the CDR of the light chain variable region of the antibody is shown as SEQ ID NO. 1-3; the amino acid sequence of the CDR of the heavy chain variable region of the neutralizing antibody is shown in SEQ ID NO. 4-6. The neutralizing antibody has no immunogenicity, good specificity, high affinity, clear components, no potential risk of pathogen pollution and the like, is widely suitable for various crowds, and is simple and efficient in preparation method and suitable for standardized production.
Description
Technical Field
The invention belongs to the field of cellular immunology, and relates to a fully human neutralizing antibody against tetanus toxin; in addition, the invention also relates to a preparation method and application of the neutralizing antibody.
Background
Tetanus (Tetanus) is an acute, lethal condition of the zoonotic nervous system caused by infection with Clostridium tetani (clostridium tetani). The clostridium tetani exists in intestinal tracts of people and livestock and in soil widely, the carrying rate of people is up to 25%, and wounds of different types and sizes can be used as portals invaded by the clostridium tetani. When people are infected, the clostridium tetani is massively propagated at the wound in the latent period under the anaerobic condition and releases toxin, and the tetanus toxin damages the central nervous system by destroying nerve cells of the human body, so that the symptoms of organism spasm, limb rigidity, opisthotonus and the like are caused, and finally, people die due to suffocation or respiratory failure. In the outbreak of war and the frequent period of natural disaster, tetanus becomes a serious public health problem, people of any age can be infected with tetanus, the death rate is 20-30%, and the death rate of serious patients and newborn babies is up to 80%.
Tetanus toxin acts rapidly and is very toxic. When patients are diagnosed as having tetanus, a large number of bacteria and toxins are generally present in the body, and the use of antibiotics cannot save the lives of the patients. The only effective way to prevent and treat tetanus is to timely inject anti-tetanus toxin antibodies to neutralize the toxin and to remove the toxin from the body mediated by the antibody receptor. In recent years, there have been increasing reports of human-murine chimeric, human and fully human monoclonal antibodies against tetanus toxin. The genetic engineering antibody prepared by transforming the fully human monoclonal antibody by utilizing the B cell screening antibody engineering technology not only eliminates the anaphylactic reaction caused by serum protein, but also overcomes the defects of the production process of human immunoglobulin, and has better development prospect. Adopting gene recombination technology to transform antibody molecules on the gene level to form a chimeric antibody, namely linking a mouse anti-variable region gene with a human IgG constant region; the humanized antibody, namely, the complementarity determining region of the variable region of the mouse antibody is transplanted into the framework region of human IgG, so that the immune response caused by the mouse antibody is further reduced; the antibody is completely humanized, does not contain any foreign gene, does not cause anaphylactic reaction, and is one of the hot spots of the current genetic engineering antibody research. However, although these reported antibodies can bind to tetanus toxin, the potency of neutralizing toxin is not high, and the problem of industrial production of antibodies has not been solved.
Therefore, there is an urgent need in the art to develop highly effective and safe neutralizing antibodies against tetanus toxin, which are fully human, using more advanced techniques, to replace commercially available serum preparations to meet human needs.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a fully human anti-tetanus toxin monoclonal neutralizing antibody, a preparation method and application thereof, wherein the antibody has no potential risks such as immunogenicity, good specificity, high affinity, clear components, pathogen pollution and the like, is widely suitable for various crowds, and is simple and efficient in preparation method and suitable for standardized production.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides an antigen-binding fragment, wherein the amino acid sequence of the CDRs in the complementarity determining region of the light chain variable region of the antigen-binding fragment is shown in SEQ ID NO. 1-3;
the amino acid sequence of the complementarity determining region CDR of the heavy chain variable region of the antigen binding fragment is shown in SEQ ID NO. 4-6.
Preferably, the amino acid sequence of the complementarity determining region CDR1 of the light chain variable region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.1 or the amino acid sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO.1 and forming the antibody or antigen-binding fragment thereof with the same or similar activity.
Preferably, the amino acid sequence of the complementarity determining region CDR1 of the light chain variable region is a homologous amino acid sequence obtained by substituting, deleting or adding not more than 20%, preferably not more than 5%, of amino acid residues in the amino acid sequence shown in SEQ ID NO.1, and the obtained amino acid sequence has tetanus toxin binding activity.
Preferably, the amino acid sequence of the complementarity determining region CDR2 of the light chain variable region is as shown in SEQ ID NO.2 or the amino acid sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO.2 and forming an antibody or antigen-binding fragment thereof having the same or similar activity.
Preferably, the amino acid sequence of the complementarity determining region CDR2 of the light chain variable region is a homologous amino acid sequence obtained by substituting, deleting or adding not more than 20%, preferably not more than 5%, of amino acid residues in the amino acid sequence shown in SEQ ID NO.2, and the obtained amino acid sequence has tetanus toxin binding activity.
Preferably, the amino acid sequence of the CDR3 of the variable region of the light chain is as shown in SEQ ID NO.3 or the amino acid sequence of the antibody or antigen-binding fragment thereof formed by substituting, deleting or adding one or more amino acids in SEQ ID NO.3 has the same or similar activity.
Preferably, the amino acid sequence of the complementarity determining region CDR3 of the light chain variable region is a homologous amino acid sequence obtained by substituting, deleting or adding not more than 20%, preferably not more than 5%, of amino acid residues in the amino acid sequence shown in SEQ ID NO.3, and the obtained amino acid sequence has tetanus toxin binding activity.
Preferably, the amino acid sequence of the CDR1 of the heavy chain variable region of the antibody or antigen-binding fragment thereof is as shown in SEQ ID No.4 or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID No.4 and forming an antibody or antigen-binding fragment thereof having the same or similar activity.
Preferably, the amino acid sequence of the complementarity determining region CDR1 of the heavy chain variable region is a homologous amino acid sequence obtained by substituting, deleting or adding not more than 20%, preferably not more than 5%, of amino acid residues in the amino acid sequence shown in SEQ ID NO.4, and the obtained amino acid sequence has tetanus toxin binding activity.
Preferably, the amino acid sequence of the complementarity determining region CDR2 of the heavy chain variable region is as shown in SEQ ID NO.5 or the amino acid sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO.5 and forming an antibody or antigen-binding fragment thereof having the same or similar activity.
Preferably, the amino acid sequence of the complementarity determining region CDR2 of the heavy chain variable region may also be a homologous amino acid sequence obtained by substituting, deleting or adding not more than 20%, preferably not more than 5%, of amino acid residues to the amino acid sequence shown in SEQ ID NO.5, and the obtained amino acid sequence has tetanus toxin-binding activity.
Preferably, the amino acid sequence of the complementarity determining region CDR3 of the heavy chain variable region is as shown in SEQ ID NO.6 or the amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO.6 and forming an antibody or antigen-binding fragment thereof having the same or similar activity.
Preferably, the amino acid sequence of the complementarity determining region CDR3 of the heavy chain variable region may also be a homologous amino acid sequence obtained by substituting, deleting or adding not more than 20%, preferably not more than 5%, of amino acid residues to the amino acid sequence shown in SEQ ID NO.6, and the obtained amino acid sequence has tetanus toxin-binding activity.
Furthermore, the amino acid sequence of the light chain variable region of the antibody or the antigen binding fragment thereof is shown in SEQ ID NO.7 or the amino acid sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO.7 and the formed antibody or the antigen binding fragment thereof has the same or similar activity.
Preferably, the variable region in the light chain has a homologous amino acid sequence obtained by substituting, deleting or adding not more than 20%, preferably not more than 5%, of amino acid residues in the amino acid sequence shown in SEQ ID NO.7, and the obtained amino acid sequence has tetanus toxin binding activity.
Furthermore, the amino acid sequence of the heavy chain variable region of the antibody or the antigen binding fragment thereof is shown as SEQ ID NO.8 or the amino acid sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO.8 and forming the antibody or the antigen binding fragment thereof with the same or similar activity.
Preferably, the amino acid sequence of the heavy chain variable region is a homologous amino acid sequence obtained by substituting, deleting or adding not more than 20%, preferably not more than 5%, of amino acid residues of the amino acid sequence shown in SEQ ID NO.8, and the obtained amino acid sequence has tetanus toxin binding activity.
Antibodies comprising conservative sequence variants of the amino acid sequences of preferred antibodies are also included within the scope of the invention. Conservative amino acid sequence variants include modifications that do not significantly alter the amino acid sequence of the fully human neutralizing antibody against tetanus toxin, binding and neutralizing properties, etc., as described herein, such as variants derived from similar amino acid substitutions, amino acid deletions, additions, etc., as are well known in the art.
The antibodies of the invention also include both human and non-human antibodies, as well as all antibodies that have the same function or are engineered and optimized as the antibodies of the invention.
The term "antibody" as used herein refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2, and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain C. The VH and VL regions can also be subdivided into regions of high denaturation, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called Framework Regions (FRs). The variable regions (VH and VL) of each heavy/light chain pair form the antibody binding sites, respectively. The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody may be of a different isotype, for example, an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype)), IgA1, IgA2, IgD, IgE, or IgM antibody.
The term "antigen-binding fragment" of an antibody as described herein refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". In particular embodiments, the antigen-binding fragment may be selected, for example, from the group consisting of: fab, Fab ', F (ab')2, Fd, Fv, dAb and Complementarity Determining Region (CDR) fragments, linear antibodies, single chain antibodies (e.g., scFv), minibodies, diabodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding capability on the polypeptide.
In a second aspect, the present invention provides a fully human neutralizing antibody against tetanus toxin comprising an antigen binding fragment according to the first aspect.
Preferably, the constant region of the antibody or antigen-binding fragment thereof comprises any one or a combination of at least two of human IgG1, IgG2, IgG3, or IgG4 constant regions, preferably human IgG1 constant regions.
In a third aspect, the present invention provides a DNA fragment comprising a nucleotide sequence encoding the antigen-binding fragment of the first aspect and/or the light chain variable region and/or the heavy chain variable region of the antibody of the second aspect.
In a fourth aspect, the present invention provides an expression vector comprising a DNA fragment according to the second aspect. In a preferred embodiment, the vector of the invention is, for example, a plasmid, cosmid, phage, cosmid, or the like.
Preferably, the expression vector comprises pcDNA3.3 vector.
In a fifth aspect, the present invention provides a host cell comprising a DNA fragment according to the second aspect and/or an expression vector according to the third aspect.
Host cells useful in the present invention include, but are not limited to, microorganisms, such as bacteria (e.coli, bacillus subtilis) transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (baculoviruses) containing antibody coding sequences; plant cell systems infected with recombinant viral expression vectors (cauliflower mosaic virus (CaMV), Tobacco Mosaic Virus (TMV)) or transformed with recombinant plasmid expression vectors containing antibody coding sequences (Ti plasmids); or a mammalian cell system (COS, CHO, BHK, 293, 3T3 cells) carrying a recombinant expression construct comprising a promoter derived from the genome of a mammalian cell (metallothionein promoter), a promoter derived from a mammalian virus (adenovirus late promoter, vaccinia virus 7.5K promoter).
In a sixth aspect, the present invention provides a method of preparing an antigen-binding fragment according to the first aspect and/or an antibody according to the second aspect, the method comprising the steps of:
(1) b cells of an immune individual are collected, and a DNA fragment of a neutralizing antibody variable region is obtained by PCR;
(2) connecting the DNA fragment in the step (1) into an expression vector, transferring into competent cells, and selecting monoclonal cells for screening after culturing;
(3) transferring the screened expression vector into host cells, culturing, collecting supernatant, separating and purifying to obtain the neutralizing antibody.
The invention utilizes the single cell RT-PCR and antibody screening technology to separate the variable region nucleotide sequence of the anti-tetanus toxin neutralizing antibody from the healthy human B cells injected with the tetanus vaccine, obtains the high-purity fully humanized neutralizing antibody protein by constructing an expression vector, and has standard and controllable preparation method and low cost.
In a seventh aspect, the present invention provides a pharmaceutical composition comprising any one of or a combination of at least two of the antigen-binding fragment of the first aspect, the antibody of the second aspect, the DNA fragment of the third aspect, the expression vector of the fourth aspect, or the host cell of the fifth aspect.
Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.
In a seventh aspect, the present invention provides a kit comprising any one or a combination of at least two of the antigen-binding fragment of the first aspect, the antibody of the second aspect, the DNA fragment of the third aspect, the expression vector of the fourth aspect, or the host cell of the fifth aspect.
Preferably, the kit further comprises a washing solution.
In an eighth aspect, the invention provides a use of the antibody of the first aspect, the DNA fragment of the third aspect, the expression vector of the fourth aspect or the host cell of the fifth aspect in the preparation of a tetanus-related drug and/or detection reagent.
In the invention, the tetanus medicine is used for preventing and/or treating tetanus.
In the present invention, "tetanus toxin" refers to a secreted protein produced by Clostridium tetani under anaerobic conditions, and is common knowledge in the art (see published sequences in NCBI GENEBANK database, etc.).
In the present invention, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population comprising identical individual antibodies, except for a few possible natural mutations. The monoclonal antibody is produced by cell strains aiming at a single antigen site with high specificity, and cross contamination does not exist; the modifier "monoclonal" is used merely to indicate the identity of the antibody, and is intended to refer to a population of substantially homogeneous antibodies, and does not imply that any particular method is required for producing the antibody.
In the present invention, the antibody includes a homolog or a modification; wherein, the homologues comprise antibody fragments obtained by transforming and optimizing the constant region or CDR partial amino acid sequence of the antibody by single-point mutation or multi-point combined mutation, CDR grafted antibody fragments, single heavy chain CDR or single light chain CDR antibody fragments obtained by transforming the constant region and the CDR amino acid sequence, CDR grafted antibodies, antibodies obtained by transforming the constant region and the CDR or scFv antibodies obtained by connecting all antibody variable regions of the single heavy chain and the single light chain containing the CDR of the antibody; the modifications include additions, deletions or modifications to the amino acid or nucleotide sequence of the antibody which still retain the ability to neutralise tetanus toxin.
In the present invention, "variable" means that the variable regions of antibodies differ in sequence to form various antibodies having a specific binding function to a specific antigen. Variability is concentrated in three segments of the Complementarity Determining Regions (CDRs) or hypervariable regions of the variable regions of the light and heavy chains of an antibody, each of which comprises four FR regions (the more conserved portions of the variable regions) in the variable regions of the native heavy and light chains, which are in a substantially β -sheet configuration, linked by three CDRs forming a connecting loop, which may form part of the β -sheet structure. The CDRs in each chain are held together tightly by the FR regions and form the antigen binding site of the antibody with the CDRs of the other chain. The constant regions are not directly involved in binding of the antibody to the antigen, but exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.
Compared with the prior art, the invention has the following beneficial effects:
(1) the neutralizing antibody of the invention has strong binding activity and affinity to tetanus toxin;
(2) the neutralizing antibody has high purity, no immunogenicity, clear components and no potential risks of pathogen pollution and the like;
(3) the method for preparing the neutralizing antibody has the advantages of controllable standard, low cost, simplicity, high efficiency and suitability for standardized production.
Drawings
FIG. 1 is an SDS-PAGE pattern of an antibody against TRN1013 according to the present invention, wherein lane 1 shows a non-reduced antibody, lane 2 shows a protein molecular weight, and lane 3 shows a reduced antibody;
FIG. 2 is a graph showing the results of the detection of the neutralizing activity of the antibody against TRN1013 and tetanus standard toxin according to the present invention;
FIG. 3 is a graph showing the affinity activity of antibodies against TRN1013 of the present invention for tetanus standard toxin;
FIG. 4 is a graph showing the results of the neutralization test of the TRN1013 antibody of the present invention at 20-fold LD50 dose in mice.
Detailed Description
To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
Those without specifying the particular techniques or conditions in the examples, according to techniques or conditions described in the literature in the field or according to the instructions of the products, such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press, 1989). The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
The term "KD" as used in the examples refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, and is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the more tight the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, the antibody is less than 10-5The dissociation equilibrium constant (KD) of M is bound to an antigen, and may be, for example, less than 10-6M、10-7M、10-8M、10-9M or 10-10M, KD was determined using a BIACORE instrument using Surface Plasmon Resonance (SPR).
Experimental Material
(1) After injecting 1500IU tetanus toxoid vaccine into healthy volunteers, 20mL blood samples are collected on 0 th day, 14 th day and 21 st day respectively for anticoagulation;
(2) reagent: tetanus vaccine, Ficoll lymphocyte isolate (Cedarlane Co.), TAT-FITC, TAT-FITC-Isotype, primer (Invitrogen), Superscript III reverse transcriptase, HotStarTaq Plus enzyme (Invitrogen, Carlsbad, CA), agarose, Tris, LB medium, ampicillin, PolyFect transfection reagent (Qiagen, Valencia, CA), FCS, DMEM medium, PBS buffer, BSA, HBsAg kit, etc.;
(3) carrier: pcDNA3.3;
(4) the strain is as follows: e.coli DH5 α;
(5) cell: 293T.
EXAMPLE 1 Single Nuclear cell isolation and plasma cell sorting
(1) Collecting 15mL blood samples from blood of healthy volunteers injected with 1500IU tetanus toxoid vaccine in anticoagulation tubes containing heparin at days 0, 14 and 21, respectively, separating with Ficoll, aspirating mononuclear cell (PBMC) layer suspension, washing 3 times with PBS, and aspirating supernatant;
(2) after cell counting, the target cell population was sorted from PBMCs using a BD FACSria flow cytometer, and single cells with intact morphology were selected and placed in a 96-well PCR (Polymerase Chain Reaction) plate (20 μ L of single cell lysate per well) so that each well contained one B cell, and stored in a freezer at-80 ℃ for use.
Example 2 isolation of antibody variable region genes from Single B cells Using RT-PCR
(1) RT-PCR: to a 96-well plate containing single B cells, 0.5. mu.M of constant region primers for heavy and light chains of different subtypes (primers designed at specific sites using a conventional method) and Superscript III reverse transcriptase were added, incubated at 37 ℃ for 1 hour, and PCR amplification was performed under the following conditions: 15min at 95 ℃; 1min at 95 ℃, 1min at 55 ℃, 1min at 72 ℃ and 30 cycles; 10min at 72 ℃; 5min at 4 ℃; the obtained product cDNA is preserved at-20 ℃;
(2) and (3) PCR: a50. mu.L system containing 5. mu.L of reverse transcription product, HotStarTaq Plus enzyme, dNTPs, and 0.5. mu.M primers specific for the heavy and light chain variable regions of different subtypes (primers were designed at specific sites using conventional methods) was PCR amplified under the following conditions: pre-denaturation at 94 ℃ for 5 min; 30s at 94 ℃, 30s at 55 ℃, 50s at 72 ℃ and 35 cycles; 7min at 72 ℃; the obtained PCR product was identified by electrophoresis on a 1% agarose gel.
The result showed that the PCR product was about 400 bp.
EXAMPLE 3 construction of expression vectors for recombinant antibodies
The PCR product of the antibody variable region gene which is identified as positive by gel electrophoresis and can be matched and paired with the light chain and the heavy chain is connected to pcDNA3.3 vector by using TA cloning method to construct an expression vector of the anti-tetanus toxin fully human neutralizing antibody, then the expression vector is transformed into DH5 alpha competent bacteria, the bacteria are cultured on a plate containing ampicillin overnight at 37 ℃, 10 single colonies are picked to carry out PCR by using specific primers, and the reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 100s, 28 cycles; extension at 72 ℃ for 5 min. Take 5. mu.L of PCR product and detect it by 1% agarose gel electrophoresis.
The results showed that among the positive transformants, transformants containing the antibody heavy and light chain genes were identified.
Example 4 antibody expression
Transforming the positive plasmid into DH5 alpha to perform mass amplification, quickly extracting recombinant plasmid, CO-transfecting 293 cells with a transfection reagent PolyFect, replacing a large amount of fresh culture medium 6-8 hours after transfection, and performing mass culture at 37 ℃ with 8% CO2After culturing for 96 hours in an incubator, cell supernatants are collected for detection.
Example 5 screening assay for expressed antibodies
Diluting tetanus toxoid by 10 times by using a coating solution, adding 100 mu L of tetanus toxoid per well into a 96-well ELISA plate, coating at 4 ℃ overnight, and sealing for 2 hours at normal temperature by using a sealing solution; adding 100 mu L of transient transfection supernatant (primary antibody) and incubating for 2 hours at 37 ℃, adding HRP/anti-His-tag (1:2000 dilution, secondary antibody) and incubating for 1 hour at 37 ℃, adding 100 mu L/hole of substrate color development liquid, standing for 5min at normal temperature in a dark place, stopping the reaction by using 2M sulfuric acid, and carrying out colorimetric detection at the wavelength of 450 nm.
EXAMPLE 6 expression and purification of antibodies
CO-transfecting 293 cells with the expression vectors of the heavy chain and the light chain of the antibody (wherein the amino acid sequence of the light chain variable region is shown as SEQ ID NO:7 and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 8) with neutralizing activity identified by a neutralization experiment, changing a large amount of fresh culture medium 6-8 hours after transfection, and carrying out 8% CO treatment at 37 DEG C2Culturing for 96 hours in an incubator; the transfection supernatant was collected, centrifuged at 4000rpm for 1 hour and subjected to protein A affinity chromatographyPurifying; the expression and purification of the antibody was examined by SDS-PAGE.
As a result, as shown in FIG. 1, the purity of the obtained protein was high, and the light chain and the heavy chain were clearly observed after melting. The 293 cell of the cotransfection expression plasmid vector successfully expresses the fully human antibody, and can effectively identify the tetanus toxoid antigen, while the 293 cell culture supernatant of the untransfected expression plasmid can not identify the tetanus toxoid antigen, namely, the transient transfection 293 successfully expresses the anti-tetanus spasm toxin fully human antibody which can specifically identify the tetanus toxoid.
Example 7 neutralizing Activity assay of monoclonal antibodies
The neutralizing activity of the expressed purified antibody was tested by the same ELISA method as mentioned before: diluting tetanus toxoid by 10 times by using a coating solution, adding 100 mu L of tetanus toxoid per well into a 96-well ELISA plate, coating at 4 ℃ overnight, and sealing for 2 hours at normal temperature by using a sealing solution; adding 100 mu L of expression and purification antibody as a primary antibody, incubating at 37 ℃ for 2h, adding HRP/anti-His-tag (diluted by 1: 2000) as a secondary antibody, incubating at 37 ℃ for 1h, adding 100 mu L/hole of substrate color development liquid, standing at normal temperature in a dark place for 5min, stopping the reaction with 2M sulfuric acid, and carrying out colorimetric detection at the wavelength of 450 nm.
As a result, as shown in FIG. 2, the TRN1013 antibody (antibody concentration of about 0.0005. mu.g/mL) diluted at a concentration of more than 50,000 times was still able to neutralize the antigen, and had a very strong neutralizing activity.
Example 8 monoclonal antibody affinity Activity assays
The antibody affinity test adopts a capture method, the buffer is HBS-EP, and a CM5 chip is firstly coupled by proteinA. The capture antibody was then diluted to a concentration of 1. mu.g/mL for a binding time of 50 s. The analyte tetanus toxoid was flowed sequentially through the chip at increasing concentrations, and signal curves were obtained, respectively. 1 cycle for each concentration, and 1 cycle after 3M MgCl2The chip was regenerated to return to the original uncaptured antibody state for 30 s. The resulting signal curves were analyzed using BiaCore X-100System software to generate a graph that measures the affinity activity of the neutralizing antibodies to tetanus toxin as provided in the examples of the present invention.
The results are shown in FIG. 3 and Table 1, for all personsThe rate of binding (ka) of the source anti-tetanus toxin neutralizing antibody was 5.51E +04Ms-1The dissociation rate (kd) reaches 6.07E-05s-1The dissociation equilibrium constant (KD) of the antibody reaches 1.10E-9M, which indicates that the antibody has high affinity to tetanus toxin; .
TABLE 1 affinity assay results for neutralizing antibodies against tetanus toxin of fully human origin tetanus toxoid
| Name of antibody | ka(1/Ms) | kd(1/s) | KD(M) |
| TRN1013 | 5.51E+04 | 6.07E-05 | 1.10E-0.9 |
Example 9 in vivo neutralization assay
(1) Determination of the median lethal dose (LD50)
Standard antitoxin and toxin dilution were prepared according to 2015 version "chinese biologicals protocol", prepared toxin was diluted with diluent in a gradient (10, 20, 40, 80, 160, 320, 640, 1280, 2560, 5120 and 10240 times), 0.2mL of mice injected, 4 mice per group, observed for 5 days, and LD50 was calculated from the experimental results, 20 times × LD50 was used in the experimental group.
(2) Determination of the potency of monoclonal antibodies
The monoclonal antibody to be detected is diluted to 100 mu g/mL (the concentration of the monoclonal antibody is more than 1mg/mL) by using a diluent, namely the concentration of the monoclonal antibody in each 0.4mL injection volume is 50 mu g/mL after the monoclonal antibody is mixed with the toxin in equal quantity.
Quantitatively mixing and sucking the diluted standard antitoxin and monoclonal antibody to be detected, respectively filling into small test tubes, adding the same amount of dilution test toxin into each tube, uniformly mixing, plugging, incubating at 37 ℃ for 1 hour, and immediately injecting.
28 healthy laboratory mice weighing 18-22g were divided into 7 groups of 4 mice each. The mixture (blank control group comprised 0.2mL Toxin (Toxin20LD50), negative control group comprised 0.2mL Toxin +0.2mL borate buffered saline (20LD50+ PAb) and negative antibody control group (20LD50+ TRN006), positive control group comprised 0.2mL Toxin +0.2mL antitoxin, experimental group comprised 0.2mL Toxin +0.2mL monoclonal antibody (20LD50+ TRN1013)) was injected subcutaneously into the abdomen of mice, each injection at 0.4 mL. The disease and death of the mice are recorded after the observation once every day in the morning and afternoon for one continuous week.
As shown in FIG. 4, the mice in the negative control group and the negative antibody control group all died within 48 hours, and the mice in the experimental group with the monoclonal antibody concentration of 20 times of lethal dose (LD50) +50 μ g/mL dose all survived, indicating that when the monoclonal antibody of the present invention is used at 50 μ g/mL dose, the titer of the standard antitoxin (10IU/mL) is equivalent, the animal can be effectively protected from the attack of tetanus toxin with lethal dose, and the protection of the monoclonal antibody is basically consistent with the protection of the standard antitoxin. Meanwhile, the actual dosage of the monoclonal antibody is far lower than that of the standard antitoxin, and the fact that the fully human antitoxic neutralizing antibody has a very strong titer effect is shown.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
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Claims (15)
1. An antigen binding fragment, wherein the amino acid sequence of complementarity determining region CDR1 of the light chain variable region of the antigen binding fragment is shown in SEQ ID NO.1, the amino acid sequence of complementarity determining region CDR2 of the light chain variable region is shown in SEQ ID NO.2, and the amino acid sequence of complementarity determining region CDR3 of the light chain variable region is shown in SEQ ID NO. 3;
the amino acid sequence of the complementarity determining region CDR1 of the heavy chain variable region of the antigen binding fragment is shown in SEQ ID NO.4, the amino acid sequence of the complementarity determining region CDR2 of the heavy chain variable region is shown in SEQ ID NO.5, and the amino acid sequence of the complementarity determining region CDR3 of the heavy chain variable region is shown in SEQ ID NO. 6.
2. The antigen-binding fragment of claim 1, wherein the light chain variable region of the antigen-binding fragment has the amino acid sequence shown in SEQ ID No. 7;
the amino acid sequence of the heavy chain variable region of the antigen binding fragment is shown as SEQ ID NO. 8.
3. A fully human neutralizing antibody against tetanus toxin comprising the antigen binding fragment of claim 1 or 2;
the constant region of the antibody comprises any one of or a combination of at least two of human IgG1, IgG2, IgG3, or IgG4 constant regions.
4. The neutralizing antibody of claim 3, wherein said antibody constant region is a human IgG1 constant region.
5. A DNA fragment comprising a nucleotide sequence encoding the antigen-binding fragment of claim 1 or 2 or the antibody of claim 3.
6. An expression vector comprising the DNA segment of claim 5.
7. The expression vector of claim 6, wherein the expression vector is pcDNA3.3 vector.
8. A host cell comprising the DNA fragment of claim 5 and/or the expression vector of claim 6 or 7.
9. The host cell of claim 8, wherein the host cell is a CHO cell or 293 cell.
10. A method of producing an antigen-binding fragment according to claim 1 or 2 and/or an antibody according to claim 3, comprising the steps of:
(1) collecting B cells of an immune individual, and obtaining a DNA fragment of an antibody variable region by PCR;
(2) connecting the DNA fragment in the step (1) into an expression vector, transferring into competent cells, and selecting monoclonal cells for screening after culturing;
(3) transferring the screened expression vector into host cells, culturing, collecting supernatant, separating and purifying to obtain the antigen binding fragment and/or the antibody.
11. A pharmaceutical composition comprising any one of the antigen-binding fragment of claim 1 or 2, the antibody of claim 3 or 4, the DNA fragment of claim 5, the expression vector of claim 6 or 7, or the host cell of claim 8 or 9, or a combination of at least two thereof.
12. The pharmaceutical composition of claim 11, further comprising any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient, or diluent.
13. A kit comprising any one of the antigen-binding fragment of claim 1 or 2, the antibody of claim 3 or 4, the DNA fragment of claim 5, the expression vector of claim 6 or 7, or the host cell of claim 8 or 9, or a combination of at least two thereof.
14. The kit of claim 13, further comprising a wash solution.
15. Use of the antigen-binding fragment of claim 1 or 2, the antibody of claim 3 or 4, the DNA fragment of claim 5, the expression vector of claim 6 or 7 or the host cell of claim 8 or 9 for the preparation of a tetanus medicament and/or detection reagent.
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| CN102875674A (en) * | 2011-10-27 | 2013-01-16 | 成都蓉生药业有限责任公司 | Anti-tetanotoxin antibody, and preparation method and application thereof |
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| CN101220096B (en) * | 2003-12-30 | 2010-09-29 | 龚小迪 | Preparation and application of humanized anti-spasmotoxin monoclone antibody |
| CN102206275B (en) * | 2011-04-27 | 2013-11-06 | 上海生物制品研究所有限责任公司 | Anti-tetanotoxin monoclonal neutral antibody, and composition and application thereof |
| EP2970484B2 (en) * | 2013-03-15 | 2022-09-21 | Amgen Inc. | Heterodimeric bispecific antibodies |
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| CN101432015A (en) * | 2006-02-15 | 2009-05-13 | 英克隆系统公司 | Functional antibodies |
| CN102875674A (en) * | 2011-10-27 | 2013-01-16 | 成都蓉生药业有限责任公司 | Anti-tetanotoxin antibody, and preparation method and application thereof |
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