FULLY HUMANIZED MONOCLONAL NEUTRALIZING ANTIBODIES FOR TETANUS TOXIDANT AND ITS USE

Description

FIELD OF TECHNOLOGY

[0001] This invention belongs to the field of Cellular Immunology and Genetic Engineering, and relates to a fully native human neutralizing monoclonal antibody against tetanus toxin and its applications.BACKGROUND OF THE INVENTION

[0002] Tetanus is an acute disease characterized by muscle spasms caused by a neurotoxin produced by Clostridium tetani after its invasion into a body through a wound and reproduction under anaerobic environment. Serious patients may die because of laryngeal spasm or severe secondary lung infection. The death rate can be as high as 20-50% among the newborns and serious patients. Every year there are about one million deaths globally caused by tetanus, mostly in undeveloped countries and regions. Clinically tetanus is categorized in four types: local tetanus, cephalic tetanus, generalized tetanus and neonatal tetanus.

[0003] C. tetani is strict anaerobic bacteria, and its spores are extremely hardy and are resistant to drought, heat and antiseptics but sensitive to iodine solution or neutral glutaraldehyde solution, and can be killed by these reagents in the short term. C. tetani can produce two kinds of exotoxins: tetanus hemolytic toxin which causes hemolysis, and tetanus spastic toxin which is the commonly known tetanus toxin.

[0004] Tetanus toxin is a protein produced and secreted by C. tetani, consisted with 1315 amino acids and with a molecular weight of 150,700Da. The estimated lethal dose for tetanus toxin is about 0.25ng / kg. Tetanus toxin is expressed as a single chain protein inside of the bacteria cell, and then is cleaved by protein enzymes into a light chain and a heavy chain coupled together by a disulfide bond. According to its in vivo functions, the tetanus toxin molecule is divided into A, B and C three regions: the light chain fragment is A fragment, N-terminal half of the heavy chain is B fragment, and the other half of heavy chain is C fragment.

[0005] Generally, the mechanism can be divided in three steps: binding, transport and action. Researches have shown that C fragment of tetanus toxin can bind to toxin receptor, which is generally believed as a ganglioside. The C fragment has the function of retrograde axonal transport to the central nervous system, and has been used to develop subunit vaccines. B fragment can form an ion channel through artificial phospholipid bilayer, transporting the active fragment of toxin into a cell. The Fragment A is a Zn protease, with proteolysis activity, and can breakdown membrane proteins involving protein-vesicular transportation of neurotransmitters. This inhibits the release of neurotransmitters and causes the continuous transmission of excitatory impulse, resulting into clinic symptom of violent spastic paralysis. However, direct isolation and purification of C fragment from tetanus toxin have many shortcomings: high toxicity of tetanus toxin, possible transmission by spores, complex processes for culture and isolation, low recovery rate and certain degree of risk.

[0006] In early days, tetanus toxoid was used for prevention and treatment of tetanus as immunoreagents, but with quite some adverse reactions, such as high sensitization. In the 60s of 20th century, developed countries in Europe and America have developed human tetanus immunoglobin (HTIG). The production of HTIG in China began in 80s, and current production of HTIG still cannot meet the demands from the market, mainly TAT (Tetanus AntiToxin) from horse serum occupies the majority of market share. Humanized HTIG has overcome the adverse reactions such as sensitization reaction caused by the clinic usage of horse serum TAT, and significantly improved the prevention and treatment of tetanus. But the human bloods are scarce sources with high cost, also with the risk of exogenous virus contamination, the industrial production and clinical use of HTIG have been largely limited.

[0007] Currently, there are reports on transforming mouse monoclonal antibody to produce genetically engineered and humanized tetanus toxin, but those are still at the stage of lab experimentation. Recently, as the rapid development of genetic engineering technology, it becomes possible to produce humanized / human antibody using genetic engineering technology. The humanized / human antibody produced via genetic engineering may reduce or eliminate the sensitization reaction caused by exnogeneic serum. It may also resolve the problems of insufficient source of human blood for human immunoglobulin production or the potential virus contamination. This has become the focus of current research.Prior art

[0008] Chin J et al. (Biologicals, Academic Press Ltd, London, vol. 31, no. 1, 1 March 2003, pages 45-53) discloses the isolation of human antibody sequences from humans vaccinated with tetanus toxin. Antibodies CH5 and CH8 have binding constants of 2.5 nM and 0.375 nM, respectively, however, CH5 has the better neutralising activity (section 3.6, 3.7). Combination of both antibodies resulted in a synergistic effect (Fig.6).

[0009] EP0562132 discloses the production of high affinity (nM), neutralising, human antibodies to tetanus toxin (see examples). Therein used are doses of toxin at L+ / 20 and L+ / 200 levels corresponding to 450MLD and 45MLD, respectively, to determine the neutralizing capacity of antibodies. Results show that ST12 antibody has the highest neutralizing capacity of 13.2 IU / 100µg IgG.

[0010] Matsuda M et al. (European Journal of Epidemiology, Springer Netherlands, Dordrecht, vol. 8, no. 1, 1 January 1992, pages 1-08) discloses the production of high affinity (nM), neutralising, human antibodies to tetanus toxin (see Tables 1 and 2); Antibody G6 has a potency of 42.9 IU / 100mcg (Table 2). Page 7, lines 11-19 discloses a method for detecting the titer of anti-tetanus antibodies. Using this method, the minimum survival dose of several anti-tetanus monoclonal antibodies G1, G2, G3, G4, and G6 prepared by the hybridoma method (Table 2) was tested, and deduced the corresponding neutralizing activity of each antibody from the corresponding minimum survival dose, by comparison with tetanus polyclonal antibody Tetanobulin (Polyethylene glycol treated human anti-tetanus immunoglobulin produced by Japan Blood Products Organization). Table 2 shows that the monoclonal antibody G6 has the minimum survival dose of 0.028µg IgG.

[0011] JP2001299360 discloses the production of high affinity, neutralising, human antibodies to tetanus toxin (see examples, Table 1. Fig.1,2), including the antibody G6 disclosed in Matsuda M et al referenced above. Although the specific titer of G6 is not described in this document, the same method as reported in Table II of Matsuda M et al is used to determine the titer of G6 (Table 1). Although G6 ScFv prolongs the death time of mice at the toxin dose of 300, 100, 30 and 10MLD, it cannot completely protect mice from death, according to the animal experimental data published in Figures 10 and 11.

[0012] Han Wang et al. (Toxins, vol. 8, no. 9, 11 September 2016, page 266) discloses the preparation and selection of human, neutralising, anti-tetanus toxin antibodies from a human scFv phage display library (Tables 1 and 2). The antibodies described in Table 4 demonstrate sub-nanomolar affinity. However, when testing the in vivo neutralizing activity of the antibodies 2-7G, 2-2D and S-4-7H, none of these antibodies can protect mice from death when 20 LD 50S (50% of the lethal dose) is used (summary and figure 7).

[0013] CN1634991 discloses anti-tetanus toxin aAb with a neutralising activity of 31,000IU / g (Example 9). Example 9 shows that all mice died at 45 hours when 0.2 mg / kg antibody was injected, and mice survived only when the amount of antibody was increased to 0.4-0.8 mg / kg.

[0014] CN108218984, CN108314730, CN108314731 and CN108623681 disclose fully human antibodies that can neutralize the tetanus toxin, which is responsible for the dangerous disease tetanus. The antibodies are developed using a specialized high-throughput method, ensuring they are safe, effective, and free from contamination, making them suitable for widespread use. These antibodies can be used to prevent and treat tetanus infections and to help detect the presence of the toxin.

[0015] CN105153305 discloses a fully human monoclonal antibody specifically designed to target and neutralize the tetanus toxin. This fully human antibody is reported to minimize risks of allergic reactions or contamination from animal sources, making it safer for patients. The antibody has a long-lasting effect in the body and can be produced in large quantities.

[0016] CN101220096 discloses a human antibody specifically designed to neutralize tetanus toxin, which is less likely to cause allergic reactions, has a longer-lasting effect, and can be produced in large quantities without the risk of animal virus contamination.

[0017] CN1634991 discloses an antibody specifically designed to fight tetanus toxin, which is derived from humans, making it less likely to cause allergic reactions and more effective in neutralizing the toxin. It offers a longer-lasting protection against tetanus, meaning fewer doses may be needed for effective treatment.Description of Invention

[0018] The invention is defined by the appended claims. References to other aspects or embodiments are for illustrative purposes only.

[0019] In order to improve the shortage of current technology, the main purpose of this invention is to provide an fully native human neutralizing monoclonal antibody against tetanus toxin and its antigen binding fragment without immunogenicity or exogenous virus contamination but with high affinity, high specificity and high efficiency, also to provide encoding sequences for the antibody, cell lines for production of the antibody, and methods and applications for using the antibody for diagnosis, prevention or therapy.

[0020] In one aspect, the invention provides a fully native human neutralizing monoclonal antibody against tetanus toxin and an antigen-binding fragment thereof, wherein the neutralizing monoclonal antibody comprises at least a heavy chain variable domain (VH) with three CDRs and at lease a light chain variable domain (VL) with three CDRs, wherein the CDR1, CDR2 and CDR3 of VH comprising an amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively; wherein the CDR1, CDR2 and CDR3 of VL comprising an amino acid sequence set forth in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, respectively.

[0021] Further, the VH of the neutralizing monoclonal antibody comprises an amino acid sequence set forth in SEQ ID NO.22, and the VL of the neutralizing monoclonal antibody comprises an amino acid sequence set forth in SEQ ID NO.26.

[0022] Furthermore, the neutralizing monoclonal antibody or the antigen-binding fragment thereof of this invention binds immune-specifically with tetanus toxin, and the neutralizing monoclonal antibody or the antigen-binding fragment thereof does not dissociate from tetanus toxin or Clostridium tetani at an equilibrium dissociation constant (KD) higher than 10 -6< M.

[0023] Preferably, the heavy chain type of the neutralizing monoclonal antibody of this invention is IgG1, IgG2, IgG3, or Ig4, and more preferably, IgG1.

[0024] Preferably, the light chain type of the neutralizing monoclonal antibody of this invention is κ or λ.

[0025] The neutralizing monoclonal antibody of this invention comprises not only a variable region but also a constant region.

[0026] Preferably, the constant region of the neutralizing monoclonal antibody of this invention is the one from any of human IgG1, IgG2, IgG3 or IgG4.

[0027] The anti-tetanus toxin antibody or its antigen binding fragment of this invention is preferable humanized antibody, comprising (not limited to) single chain Fv (scFv), Fab, Fab', F(ab')2, Fv, dsFv, diabody, Fd or Fd' fragment.

[0028] The neutralizing monoclonal antibody or the antigen-binding fragment thereof of this invention also comprises a peptide adaptor, and preferably, the peptide adaptor comprises about 1-50 amino acids.

[0029] In another aspect, this invention provides a conjugate, and the conjugate which consists of the above described neutralizing monoclonal antibody or the antigen-binding fragment thereof of this invention, and a labeling conjugated to the neutralizing monoclonal antibody or the antigen-binding fragment thereof of this invention, such as testable part and reagents. The labeling is directly conjugated covalently or non-covalently to the neutralizing monoclonal antibody or the antigen-binding fragment thereof of this invention. Also, the labeling can be conjugated through one or more adaptors to the neutralizing monoclonal antibody or the antigen-binding fragment thereof of this invention. The technics for conjugating labeling to the neutralizing monoclonal antibody or the antigen-binding fragment thereof of this invention are well known to technicians of this field. The testable part and / or reagents of the labeling include free enzymes, prosthetic groups, fluorescent materials, bioluminescent materials, radioactive materials, positron emitting materials, and non-radioactive paramagnetic metal ions.

[0030] In another aspect, this invention provides a composition, comprising the above described neutralizing monoclonal antibody or its antigen-binding fragment, and a chemical or biological labeling covalently linked to the above described neutralizing monoclonal antibody or its antigen-binding fragment.

[0031] Preferably, said chemical labeling including isotopic labeling, immunotoxin labeling, or chemical drug labeling; and said biological labeling including biotin labeling, avidin labeling, enzyme labeling, fluorescent labeling, or electron transfer agent labeling.

[0032] In another aspect, this invention provides a conjugate, comprising the above described neutralizing monoclonal antibody or its antigen-binding fragment, and / or above described conjugate, and / or above described composition coupled to a solid or semi-solid medium.

[0033] The examples of solid or semi-solid medium include enzyme-coated plate (ELISA plate), magnetic or non-magnetic immunosphere or immunogranules, and cell dispersing agent. Examples of the cell dispersing agent includes poly-vinyl-pyrrolidone-coated colloidal silicon dioxide, polysaccharide and sodium amidotrizoate or its derivatives.

[0034] In another aspect, this invention provides a combination of compositions comprising the above described neutralizing monoclonal antibody or its antigen-binding fragment, and / or above described conjugate, and / or above described composition, and / or above described conjugate.

[0035] The combination of compositions includes drug combination of compositions and diagnostic combination of compositions.

[0036] The above described drug combination of compositions also includes acceptable pharmaceutic adjuvant. The acceptable pharmaceutic adjuvants are those adjuvant or diluent agent with little significant irritation to body and no effect to eliminate the biological activities of pharmaceutic compounds. Examples include saline, sterile water, Ringer's solution, buffered saline, glucose solution, maltodextrin solution, glycerin, ethanol, or a mixture of two or more thereof. If necessary, the drug combination of compositions described in this invention may include other regularly used additives, such as antioxidants, buffers, bacteriostatic, dispersants, surfactants, adhesives and lubricants. It can also be made into different dosage forms, such as injectable preparations like solutions, suspensions or emulsions, pills, capsules, granules or tablet.

[0037] The drug combination of compositions must be sterile and stable in production or storage. When producing sterile powder for injectable solutions, the preferred method is vacuum drying or frozen drying. Vacuum drying or frozen drying can transform sterilized filtered solutions into powders while keeping the activity of the pharmaceutic compounds. Optionally, pharmaceutically acceptable excipients may be added into a solution of the drug combination of composition of this invent to generate injectable unit dosage form. Preferably, the pharmaceutically acceptable excipients used in this invention are suitable for high drug concentration, are able to keep adequate fluidity, and if necessary, are able to delay drug absorption.

[0038] Several factors influence the choices of the best route of administration, including the physical and chemical properties of the active ingredients of the drug combination of compositions, the seriousness and urgency of symptoms, and the relationships between the blood concentration of the active ingredients and expected drug effects. For example, adjuvant may be added during the preparation of the neutralizing monoclonal antibody of this invention, which may protect the antibody from fast release (such as release-control adjuvant), such adjuvant may include implants, patches, or microcapsules. Polymers that is biodegradable and biocompatible can be used in this invention, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyester or polylactide. Furthermore, the neutralizing monoclonal antibody may mix with compounds or materials that can protect the antibody from loss of bioactivity, or administrated together with such compounds or materials. For example, the neutralizing monoclonal antibody may be administrated together with appropriate adjuvant such as liposomes or diluents.

[0039] The route of administration of drug combination of compositions of this invention may be oral or parenteral administration. The preferred route is intravenous injection, but not limited to it.

[0040] The oral dosage form can be tablet, lozenge, sugar lozenge, water-based or oil-based suspension, power, granule, emulsion, hard capsule, soft capsule, syrup, pill, sugar-coated pill, liquid, gel, or ointment. These may also contain some excipients, including but not limited to, granulating agent, disintegrating chemical, adhesive, lubricant, preservative, colorant, blending agents or sweetener, vegetable oil or mineral oil, humectant, and thickening agent.

[0041] The parenteral dosage form may be water based or non-water based sterile isotonic solution for injection or perfusion, or suspension. Said solution or suspension may be agents that are not toxic to subjects at an administrative concentration and dosage, such as 1, 3-butanediol, Ringer's solution, Hank's solution, isotonic sodium chloride solution, oil, fatty acid, localized anesthetic, preservative, buffer solution, agent increasing viscosity or solubility, water soluble antioxidant, oil soluble antioxidant and metal chelating agent.

[0042] The said drug combination compositions may also include one or more other therapeutic agents, such as antibody, small molecular weight compounds, organic compounds, inorganic compounds, enzymes, or polynucleotides.

[0043] In another aspect, this invention provides a testing product, comprising the above described neutralizing monoclonal antibody or its antigen-binding fragment, and / or the conjugate, and / or the composition, and / or the conjugate, and / or the combination of compositions.

[0044] The testing product of this invention may be used to detect tetanus toxin or Clostridium tetani infection. Specifically, the testing product of this invention measures the level of tetanus toxin in fluid, cell or tissue sample through antibody. At the same time, the testing product provided in the examples of this invention is able to compare the measured tetanus toxin level with the control level. A higher measured tetanus toxin level than control level indicates infection by tetanus toxin. Preferably, the cell or tissue samples are from human subject, and the samples may include blood, urine, saliva, lavage sample or lymphoid tissue.

[0045] In another aspect, this invention provides a DNA sequence encoding the above described neutralizing monoclonal antibody or the antigen-binding fragment thereof.

[0046] Said DNA sequence may be obtained using any known methods of this field of technology. For example, if the nucleotide sequence of the antibody is known, said antibody encoding DNA sequence may be assembled with chemically synthesized oligonucleotides.

[0047] Or, said antibody encoding DNA molecule may be generated from nucleotides from proper source. If it is unable to obtain a clone of said antibody encoding DNA molecules, but the sequence of said antibody is known, then the immunoglobulin DNA molecules may be chemically synthesized, or may be amplified by PCR using synthesized 3'- an 5'- primers capable of hybridizing with said sequence; or to obtain said encoding DNA molecules using cloning with an oligonucleotide probe which is specific for the said encoding sequence, for example, to isolate encoding cDNA clone from a cDNA library. Then the amplified DNA molecules by PCR may be cloned into a replicable cloning vector using any known methods of this field.

[0048] In another aspect, this invention provides a recombinant expression vector, comprising above described DNA molecule.

[0049] The expression vector for construction of said recombinant expression vector may include but not limited to MarEx expression vector by Celltrion Inc., market widely available pCDNA vector, F, R1, RP1, Col, pBR322,ToL,Ti vector, cosmid vector, phage such as l phage, M13 phage, Mu phage, P1 phage, P22 phage, Qm phage, T-even phage, T2 phage, T4 phage, T7 phage, plant virus vectors. Any of the expression vectors known to the technicians in the field may be used, and selection of the expression vectors depends on properties of selected host cells. The expression vector may be introduced into a host cell by (but not limited to) for example, calcium phosphate transfection, virus infection, DEAE-dextran induced transfection, liposome transfection or electroporation transfection. Any technicians in this field may choose or use a method suitable to introduce any expression vectors into any host cells. Preferably, said expression vector contains one or more selection marker, but not limited by this, a vector without selection marker may also be used. The choice for selection of markers depends on the selected host cell as well known by the technicians in this field, but this is not critical for this invention.

[0050] In order to benefit the purification of the DNA molecules of this invention, a tag sequence may be inserted into the expression vector. Examples of tag sequences include but not limited to, six histidine tag, hemagglutinin tag, myc tag or FLAG tag. Any tag that is known to the technicians of this field to benefit the purification may be used in this invention.

[0051] In another aspect, this invention provides a recombinant cell, said recombinant cell results from transformation or transfection of a host cell with above described expression vector.

[0052] Any cell that is known to the technicians of this field may be used as a host cell. The host cell in this invention may include but not limited to, microorganism, such as bacterial transformed with recombinant phage DNA containing antibody encoding sequence, plasmid DNA or cosmid DNA (i.e. E.coli, B. subtilis), yeast transformed with a recombinant yeast expression containing antibody encoding sequence such as Saccharomyces and Pichia, insect cell system infected with a recombinant virus expression vector such as baculovirus, plant cell system infected with a recombinant virus expression vector (such as CaMV or TMV) containing antibody encoding sequence, or a mammal cell system (such as COS, CHO, BHK, 293, 3T3 cell) transfected with a recombinant expression vector with a promoter from mammal cell genome (such as metallothionein promoter) or from a mammal cell virus (such as adenoviral late promoter, or poxvirus 7.5 K promoter).

[0053] In another aspect, this invention provides the use of the neutralizing monoclonal antibody or the antigen-binding fragment thereof for detection of the existence or the level of tetanus toxin or Clostridium tetani in a sample. In a preferable example, the neutralizing monoclonal antibody or the antigen-binding fragment thereof comprises detectable labeling. In another preferable example, there is used a detectable labeling coupled to a second antibody to detect the neutralizing monoclonal antibody or the antigen-binding fragment thereof of this invention. The said use may be for diagnostic purpose or non-diagnostic purpose (for example the sample may be cell sample of sample from a patient).

[0054] In another aspect, this invention relates to the diagnosis of a subject infected with tetanus toxin or Clostridium tetani, including detection of the existence of tetanus toxin or Clostridium tetani in a subject's sample. In a preferable example, the neutralizing monoclonal antibody or the antigen-binding fragment thereof comprises detectable labeling. In another preferable example, there is used a detectable labeling coupled second antibody to detect the neutralizing monoclonal antibody or the antigen-binding fragment thereof of this invention.

[0055] In another aspect, this invention provides for the use of the neutralizing monoclonal antibody or the antigen-binding fragment thereof of this invention in a method to neutralize the toxicity of tetanus toxin in a sample, including mixing the tetanus toxin containing sample withthe neutralizing monoclonal antibody or the antigen-binding fragment thereof of this invention. The said method may be for diagnostic purpose or non-diagnostic purpose (for example the sample may be cell sample of sample from a patient).

[0056] In another aspect, this invention provides an application of the above described neutralizing monoclonal antibody or its antigen-binding fragment, and / or the conjugate, and / or the composition, and / or the conjugate, and / or the combination of compositions for production of testing product for detection of tetanus toxin or Clostridium tetani infection.

[0057] Preferably, said testing product is a detection kit, an ELISA plate, or a chip.

[0058] In another aspect, this invention provides the use of above described neutralizing monoclonal antibody or its antigen-binding fragment, and / or the conjugate, and / or the composition, and / or the conjugate, and / or the combination of compositions for the manufacture of drugs or medications, for prevention or treatment of diseases caused by tetanus toxin or Clostridium tetani infection.

[0059] In another aspect, this invention provides of the use of above described DNA molecule, recombinant expression vector, recombinant cell for manufacture of drugs or medications for prevention or treatment of diseases caused by tetanus toxin or Clostridium tetani infection.

[0060] As used in this Description, the term "antibody" is defined as a protein molecule acting as a receptor specifically recognizing an antigen, including immunoglobulin reacting with specific antigen, including the whole antibody, dimer, trimer o polymer of antibody, bispecific antibody, chimeric antibody, recombinant or engineered antibody, and a fragment thereof, so long as the fragment contains the antigen binding region.

[0061] A whole antibody may belong a type of IgA, IgD, IgE, IgM and IgG, and IgG may include IgG1, IgG2, IgG3 and IgG4 subtype. Antibody fragment is the region with antigen binding function, including Fab, Fab', F(ab') 2 and Fv. A Fab fragment contains a light chain variable region and a heavy chain variable region, light chain constant region and first heavy chain constant region (CH1 domain), also an antigen biding site. Fab' differs from Fab for its hinge region. The c-terminus of heavy chain CH1 domain contains at least a cysteine residue. F(ab') 2 antibody is resulted from the formation of disulfide bond between the cysteine residues located in the hinge region of Fab' fragment. Fv (variable fragment) is the least antibody fragment only containing a heavy chain variable region and a light chain variable region. Double chain Fv (dsFv) contains a heavy chain variable region coupled to a light chain variable region through disulfide bond. Single chain Fv (scFv) usually contains a heavy chain variable region coupled to a light chain variable region through a peptide adaptor. Such antibody fragment may result from proteases lysis (such as using papain to zymolyze a whole antibody to obtain F(ab') 2 fragment). Preferably, an antibody fragment may be constructed using recombinant genetic engineer technology.

[0062] The term "neutralizing monoclonal antibody" is defined as a monoclonal antibody with neutralizing activity. Monoclonal antibodies are antibodies with a single molecular composition with unique binding specificity and affinity to a specific epitope. Typically, immunoglobulin contains a heavy chain and a light chain. Both heavy chain and light chain contain a constant region and a variable region (a region is also called a domain). Each light chain or heavy chain contains four framework regions, separated by three variable regions, also called complementarity determining regions (thereafter, "CDR"). CDR is mainly responsible for binding to antigen epitope. The CDRs in each chain are usually CDR1, CDR2 and CDR3, as numbered successively from N-terminus.

[0063] The neutralizing monoclonal antibody of this invention also includes its functional variants, said functional variants are able to bind tetanus toxin or its fragment, and have the neutralizing activity against the said subtype or fragment of tetanus toxin.

[0064] More specifically, such functional variants may include derivatives having basically the similar primary sequence with the neutralizing monoclonal antibody of this invention but with chemical or biochemical modifications that are not found in the neutralizing monoclonal antibody of this invention. Said modifications may include acetylation, acylation, covalently coupling with nucleotide or nucleotide derivative, covalently coupling with lipid or lipid derivative, crosslinking, disulfide bond formation, glycosylation, hydroxylation, methylation, oxidation, polyethylene glycosylation, protein hydrolysis and phosphorylation.

[0065] Optionally, the functional variants may include the following neutralizing monoclonal antibodies: an amino acid sequence with one or more replacement, insertion, or deletion, or the combination thereof, of amino acids as compared with the amino acid sequence of parent neutralizing monoclonal antibody. Further, the functional variants may have truncated amino acid sequence at its N-terminal end or C-terminal end or both ends. The functional variants may have the same or different, higher or lower affinity as compared with the parent neutralizing monoclonal antibody, but still bind tetanus toxin or its fragment. For example, a functional variant of this invention may have increased or reduced binding affinity with tetanus toxin or its fragment.

[0066] Preferably, the amino acid sequence that may be modified include but not limited to framework region, highly variable region, especially variable region of CDR3. In general, a light chain and a heavy chain contain three highly variable regions (including three CDRs) and more conserved regions (the so-called framework region, FR). A highly variable region contains amino acid residues from CDR and highly variable loop. Technicians of this field may use well-known computer algorithms, such as Gap or Bestfit to compare amino acid sequences, to define the same or similar residues. Some generally used methods in molecular biology (such as PCR, oligonucleotide-directed mutagenesis, site-directed mutagenesis) may be employed to modify parent neutralizing monoclonal antibody or its fragment, or another option is to generate functional variants by organic synthesis.

[0067] The technicians of this field will also understand that the modifications of amino acid sequence of anti-tetanus toxin antibodies covered in this invention, and as an example, modified binding affinity or other biological properties of the antibodies. The amino acid variants of anti-tetanus toxin antibody may be produced with introducing appropriate nucleotide changes to the anti-tetanus toxin antibody encoding sequence, or by peptide synthesis. Such modifications may include (for example) replacement, deletion or insertion of residues. The deletion, insertion, replacement of residues, or combination thereof, complete the final construction of functional variants, the restrictions of which depends on the requested functional characters of the final constructions. Changes of amino acid may also change the post-translation process, such as the numbers or sites of glycosylation.

[0068] The advantages and beneficial effects of this invention are the followings: This invention develops fully native human neutralizing monoclonal antibodies against tetanus toxin through a high throughput fully native human monoclonal antibody systematic development platform.

[0069] The fully native human neutralizing monoclonal antibodies against tetanus toxin developed in this invention is applicable to the prevention and treatment of Clostridium tetani infection.

[0070] The fully native human neutralizing monoclonal antibodies against tetanus toxin developed in this invention is applicable to the detection of Clostridium tetani infection.

[0071] The fully native human neutralizing monoclonal antibodies developed in the invention have a high affinity with tetanus toxin, and high neutralizing activities, safe with high effectiveness, free of exogenous virus contamination, and can be widely used in all types of human groups with high industrial utilities.Description of Figures

[0072] Figure 1 shows the detection of the expression and purification of TRN0010 antibody using SDS-PAGE and Western Blot, and A for SDS-PAGE, B for Western Blot. Figure 2 shows the detection of the expression and purification of TRN0012 antibody using SDS-PAGE and Western Blot, and A for SDS-PAGE, B for Western Blot. Figure 3 shows the detection of the expression and purification of TRN0011 antibody using SDS-PAGE and Western Blot, and A for SDS-PAGE, B for Western Blot. Figure 4 shows the detection of the expression and purification of TRN1015 antibody using SDS-PAGE and Western Blot, and A for SDS-PAGE, B for Western Blot. Figure 5 shows the results for determination of neutralizing activity of TRN0010 antibody using ELISA. Figure 6 shows the results for determination of neutralizing activity of TRN0012 antibody using ELISA. Figure 7 shows the results for determination of neutralizing activity of TRN0011 antibody using ELISA. Figure 8 shows the results for determination of neutralizing activity of TRN1015 antibody using ELISA. Figure 9 shows the results for measuring affinity of TRN0010 antibody. Figure 10 shows the results for measuring affinity of TRN0012 antibody. Figure 11 shows the results for measuring affinity of TRN0011 antibody. Figure 12 shows the results for measuring affinity of TRN1015 antibody. Figure 13 shows the results for protection effects on animals with TRN0010 antibody. Figure 14 shows the results for protection effects on animals with TRN0012 antibody. Figure 15 shows the results for protection effects on animals with TRN0011 antibody. Figure 16 shows the results for protection effects of TRN1015 antibody against 20 times dose of LD50 tetanus toxin. Figure 17 shows the results for protection effects of TRN1015 antibody against 60 times dose of LD50 tetanus toxin. Specific Method of Use

[0073] The followings further describe this invention in detail with figures and examples. The following \examples are only for further description on this invention, and by no means to limit the scope of this invention. Some of the conditions and methods may not disclosed in detail, those conditions and methods are generally described in Molecular Cloning, a Lab Manual by Sambrook et al. of New York Cold Spring Harbor Laboratory Press, 1989, or that recommended by manufacturers.

[0074] Example 1. Preparation of al human neutralizing monoclonal antibody against tetanus toxin. 1. Isolation of Cells. Blood samples were collected from healthy volunteers injected with 1500IU tetanus toxin, then mononuclear cells (PBMC) were isolated using Ficoll. After cell counts obtained, plasma cells were isolated by FACS using BD FACSria. Intact single cells were placed into 96-well PCR plates, with one single memory B cell per well and then kept in -80°C freezer for future usage. 2. Isolation of genes for variable regions of antibody

[0075] 0.5 mM primers for each subtype constant region of heavy chain and light chain and Superscript III reverse transcriptase were added into the 96-well plate with single B cell, incubated at 37°C for an hour. PCR was performed under the following parameters: 93°C 15 min; 95°C 1 min, 55°C 1 min, 72°C 1 min, 30 cycles; 72°C 10 min; 4°C 5 min. cDNA products kept at -20°C.

[0076] Using above cDNA as template to amplify anti-tetanus toxin fully native human antibody gene. Mixing of 5ml product of RT reaction, HotStar Taq Plus polymerase (Invitrogen, Carlsbad, CA), dNTPs, and 0.5 mm of specific primers for each subtype heavy chain and light chain. Reaction conditions: pre-denaturation at 94°C 5 min, followed by 35 PCR cycles with the conditions as 94°C x 30s, 55°C x 30s, 72°C x 50s, and at last extension at 72°C for 7 min. The PCR products were identified with 1% agarose gel electrophoresis

[0077] 3. Construction of eukaryotic expression vector for fully native human monoclonal antibody against tetanus toxin. (1) taking 2ml PCR product on 1% agarose gel electrophoresis for detection PCR results. Positive PCR product with complementary pairing between genes for variable regions of heavy chain and light chain were inserted into pcDNA3.3(+ / -) expression vector (from Invitrogen) using TA cloning method to construct fully native human anti-tetanus toxin monoclonal antibody expression vector (2) transforming DH5a competent cells with the above expression vector, culturing the transformed cells on ampicillin containing plate at 37°C overnight. (3) picking 10 single colonies for PCR confirmation using specific primers, reaction conditions: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 1min40s, 28 cycles, and at last extension at 72°C for 5 min. (4) taking 5 ml PCR product for 1% agarose gel electrophoresis confirmation, and positive transformants were identified.

[0078] Results showed that the sequence of constructed anti-tetanus toxin monoclonal antibody heavy / light chain recombinant expression vector was correct.4. Expression and identification of anti-tetanus toxin fully native human neutralizing monoclonal antibody

[0079] Amplifying the plasmids expressing positive antibody heavy and light chain genes in E coli DH5a, and rapidly isolating recombinant plasmids. 293 cells were transfected with anti-tetanus toxin fully native human neutralizing monoclonal antibody expression vector by PolyFect DNA transfection kit according to the instructions from manufacturer. Non-transfected 293 cells served as controls. After 96 hours culture, using ELISA with HPR labeled sheep anti human IgG to detect the expression of anti-tetanus toxin fully native human neutralizing monoclonal antibody and its specific recognition of tetanus vaccine (antigen).

[0080] Using tetanus vaccine as antigen and coating a 96 well ELISA plate with 10 times diluted antigen, 100 ml per well at 4°C overnight. Blocking with blocking solutions at room temperature for 2 hours, then incubating with 100 ml transient expression supernatant as primary antibody at 37°C for 2 hours, incubating with 1:2000 diluted HRP / anti-His-tag as secondary antibody at 37°C for 1h, adding substance color liquid 100 ml per well, placed away from light at room temperature for 5 min, then terminating the reaction with 2M sulfuric acid, and detect at 450 nm wavelength.

[0081] Results showed that 293 cells transfected with expression vector plasmids successfully expressed fully native human antibody, and the antibody was able to specifically recognize tetanus vaccine (antigen), while the cultural supernatant from untransfected 293 cells was not able to recognize tetanus vaccine (antigen), therefore l transiently transfected 293 cells successfully expressed fully native human anti-tetanus toxin antibody which specifically recognized tetanus vaccine (antigen).

[0082] 5. Production and purification of fully native human anti-tetanus neutralizing monoclonal antibody.

[0083] Co-transfecting 293 cells with positively identified with neutralizing activity and numbered TRN0010 antibody (comparative embodiment) heavy chain and light chain expression vector (the amino acid sequence of variable region of heavy chain is set forth in SEQ ID NO:7 and the amino acid sequence of variable region of light chain is set forth in SEQ ID NO:8).

[0084] Co-transfecting 293 cells with positively identified with neutralizing activity and numbered TRN0012 antibody (comparative embodiment) heavy chain and light chain expression vector (the amino acid sequence of variable region of heavy chain is set forth in SEQ ID NO: 17 and the amino acid sequence of variable region of light chain is set forth in SEQ ID NO:18).

[0085] Co-transfecting 293 cells with positively identified with neutralizing activity and numbered TRN0011 antibody heavy chain and light chain expression vector (the amino acid sequence of variable region of heavy chain is set forth in SEQ ID NO:22 and the amino acid sequence of variable region of light chain is set forth in SEQ ID NO:26).

[0086] Co-transfecting 293 cells with positively identified with neutralizing activity and numbered TRN1015 antibody (comparative embodiment) heavy chain and light chain expression vector (the amino acid sequence of variable region of heavy chain is set forth in SEQ ID NO:33 and the amino acid sequence of variable region of light chain is set forth in SEQ ID NO:34).

[0087] Replacing with fresh culture medium 6-8 hours after transfection, and continuously incubating at 37°C in a 8% CO2 incubator for 96 hours. Collecting transfection supernatant and centrifuging at 4000rpm for 1 hour, then purifying with Protein A affinity chromatography. Identifying the expression and purity of antibody using SDS-PAGE and Western Blot. As shown in Figures 1-4, relatively pure protein was obtained, and the heavy chain and light chain of antibody was clearly identifiable after melting. Note: Lane 1 of Figures 1-4A and Figures 1-4B representing non-melting protein, Lane 2 of Figures 1-4A and Figures 1-4B representing protein markers and Lane 3 1 of Figures 1-4A and Figures 1-4B representing melting protein.Example 2. Detection of neutralizing activities of neutralizing monoclonal antibody

[0088] Using tetanus vaccine as antigen and coating a 96 well ELISA plate with 10 times diluted antigen, 100 ml per well at 4°C overnight. Blocking with blocking solutions at room temperature for 2 hours, then incubating with serial dilution of purified antibody as primary antibody at 37°C for 2 hours, incubating with 1:2000 diluted HRP / anti-His-tag as secondary antibody at 37°C for 1h, adding substance color liquid 100 ml per well, placed away from light at room temperature for 5 min, then terminating the reaction with 2M sulfuric acid, and detect at 450 nm wavelength.

[0089] Results are shown in Figures 5-8. After dilution of purified antibody to not lower than 10,000 times, the concentration of antibody was as low as 0.0002mg / ml. The diluted antibody still was able to bind to antigen, showing its strong binding activities and neutralizing activities.Example 3. Measuring affinity of neutralizing monoclonal antibody

[0090] Coupled capturing molecules to CM5 chip and then activating Dextran surface of the chip. The amount of coupling was controlled by sampling time, and lastly capturing ligands with captured molecules. The prepared fully native human anti-tetanus toxin neutralizing monoclonal antibody was used as ligand, and HS-EB buffer diluted tetanus toxin was used as analyte. Analyte with increased concentration passed through the chip orderly, and signals were recorded respectively. Each concentration of analyte reacted with the chip for one cycle and after each cycle, the chip was regenerated with 10mmol / L glycine-HCl solution to return to its original status with no binding antigen. Kinetic analysis of binding affinity of monoclonal antibody to tetanus toxin was performed using BiaCore X-100 system software.

[0091] Results are shown in Figure 9-12 and Table 1, the equilibrium dissociation constant of neutralizing monoclonal antibody of this invention to tetanus toxin is lower than 10 -9< mol, indicating it has very high affinity activities. Table 1 Detecting results of affinity of fully native human anti-tetanus toxin neutralizing monoclonal antibodyAntibodyKa (l / Ms)Kd(l / s)KD(M)TRN0010-comparative4.15E+041.53E-043.68E-09TRN0012-comparative8.06E+043.28E-044.07E-09TRN00114.27E+043.87E-059.05E-09TRN1015-comparative3.88E+049.40E-052.43E-09 Example 4. Animal protection experiments of anti-tetanus toxin neutralizing monoclonal antibody(1) Dilution of anti-tetanus toxin monoclonal antibody for testing

[0092] The monoclonal antibody was diluted to 100 mg / ml, then a serial of 3 times dilution was made, reserved for testing.(2) Dilution of standard antitoxin

[0093] Mixing standard antitoxin saline solution with equal volume neutral glycerin (sterilized at 116°C for 10 min) then diluting into 0.5IU / ml solution, that gave IU / 10 of every 0.4ml injection after mixing with equal volume of toxin solution. Each uptake of standard antitoxin solution should not less than 0.5 ml.(3) Dilution of toxin

[0094] Dissolving freeze-dried powder toxin from Chinese National Biotesting Lab in saline and mixing with equal volume of neutral glycerin (sterilized at 116°C for 10 min), then diluting toxin solution with diluent to working concentration.(4) Determination of median lethal dose (LD50)

[0095] Diluting prepared toxin solution to 10 2< , 10 3< , 10 4< , 10 5< , 10 6< , and 10 7< , for at least 2 ml for each dilution level. Taking 0.2 ml and injecting into mice, 4 for each group, and observing for 5 days. Calculating to obtain LD50 using experimental data. The experimental group should use an amount of toxin equal to 20 times or even 60 times of LD50.(5) Determination of antibody titer

[0096] To take equal volume of diluted antitoxin and serially diluted antibody into small test tubes, to mix with equal volume of diluted toxin (20 times or 60 times of LD50). After mixing evenly, to seal with test tube plugs, reacting for 1 hour at 37°C and then to inject immediately in the mice. 140 healthy mice were taken and grouped into 4 mice per group for such experiment. Subcutaneously injecting 0.4ml above mixtures at abdomen of 18-22g mice (for negative control groups, injecting 0.2 ml toxin + 0.2 ml borate buffered saline; for positive control groups, injecting 0.2 ml toxin + 0.2 ml antitoxin; and for experimental groups, injecting 0.2 ml toxin + 0.2 ml monoclonal antibody). Observing the status of mice twice every day, one in the morning and one in the afternoon, recording the illness and death of mice.

[0097] Results are shown in Figures 13-16. When attacked with 20 times LD50 tetanus toxin, all mice in negative control groups were dead, except for the mice in experimental groups with 1.85µg / ml TRN0010 antibody died after 7 days, all mice in all other experimental groups (0.62 µg / ml, 5.56 µg / ml, 16.67 µg / ml, 50µg / ml) and all mice in positive control groups survived. This indicate low dose monoclonal antibody as low as 0.62 µg / ml was equivalent to 10UI / ml standard antitoxin. The monoclonal antibody is able to protect animals from the lethal dose attack of tetanus toxin, and has about the same protective activity as the standard antitoxin. The practical dose of monoclonal antibody of this invention was far lower than that of standard antitoxin, indicating its effects were superior to that of antitoxin. The results indicate that the monoclonal antibody of this invention is capable to neutralize the toxin inside the mice's bodies, to protect the mice and has in vivo activities.

[0098] As shown in Figure 17, when under a higher dose tetanus toxin attack (60 times LD50), the mice in negative control groups died within 24 hours, the mice in low dose experimental groups (0.62µg / ml, 1.85 µg / ml) died within 7 days, the mice in mid and high dose experimental groups (5.56 µg / ml, 16.67µg / ml and 50µg / ml) all survived within 7 days, indicating the monoclonal antibody of this invention was able to defend high dose toxin attack, also indicating the monoclonal antibody possesses extremely strong in vivo neutralizing activities.

Claims

1. A fully native human neutralizing monoclonal antibody against tetanus toxin or an antigen-binding fragment thereof, characterized in that, the neutralizing monoclonal antibody comprises a heavy chain variable domain (VH) having three CDRs and a light chain variable domain (VL) having three CDRs, wherein the amino acid sequence of the VH of the neutralizing monoclonal antibody is set forth in SEQ ID NO: 22, and the amino acid sequence of the VL is set forth in SEQ ID NO: 26.

2. A DNA molecule encoding the neutralizing monoclonal antibody or the antigen-binding fragment thereof of claim 1.

3. A recombinant expression vector, wherein said recombinant expression vector comprises the DNA molecule of claim 2.

4. A recombinant cell, wherein said recombinant cell is obtained by transformation or transfection of a host cell with the recombinant expression vector of claim 3.

5. A conjugate, characterized in that, said conjugate is formed by conjugating the neutralizing monoclonal antibody or the antigen-binding fragment thereof of claim 1, and a chemical or biological labeling linking covalently to the neutralizing monoclonal antibody or the antigen-binding fragment thereof preferably, said chemical labeling including isotopic labeling, immunotoxin labeling, and chemical drug labeling, and said biological labeling including biotin labeling, avidin labeling, enzyme labeling, fluorescent labeling, and electron transfer agent labeling6. The conjugate of claim 5, coupled to a solid or semi-solid medium.

7. A composition or kit, comprising the neutralizing monoclonal antibody or the antigen-binding fragment thereof of claim 1, and / or the conjugate of claim 5, and an acceptable pharmaceutical adjuvant.

8. An ELISA plate, or a chip, which comprises the neutralizing monoclonal antibody or the antigen-binding fragment thereof of claim 1, and / or the conjugate of claim 5, and / or the composition of claim 7.

9. The in vitro use of the neutralizing monoclonal antibody or the antigen-binding fragment thereof of claim 1: (1) for detecting the existence or the level of tetanus toxin or Clostridium tetani in a sample; (2) for detecting whether a subject is infected with tetanus toxin or Clostridium tetani; or (3) for neutralizing the toxicity of tetanus toxin in a sample.

10. The neutralizing monoclonal antibody or the antigen-binding fragment thereof of claim 1, and / or the conjugate of claim 5, and / or the composition of claim 7, for use in detecting tetanus toxin or Clostridium tetani infection.

11. The neutralizing monoclonal antibody or the antigen-binding fragment thereof of claim 1, and / or the conjugate of claim 5, and / or the composition of claim 7, or the DNA molecule of claim 2, or the recombinant expression vector of claim 3, or the recombination cell of claim 4, for use in the prevention or treatment of a disease induced by Clostridium tetani infection.