RU2815280C1 - Tetanus toxin antibody and use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described is a group of inventions comprising a monoclonal antibody that binds to tetanus toxin, use of a monoclonal antibody in the manufacture of a drug for preventing or treating tetanus and a method for preventing or treating tetanus, involving administering said monoclonal antibody to a subject. In one embodiment, the monoclonal antibody comprises a heavy chain variable region amino acid sequence selected from SEQ ID NO: 15–17, and the amino acid sequence of the light chain variable region SEQ ID NO: 18.

EFFECT: invention extends the range of tetanus toxin-binding agents.

4 cl, 9 dwg, 2 tbl, 11 ex

Description

CROSS REFERENCE TO RELATED APPLICATION

This invention application claims priority to Chinese Patent Application No. 202011128926.0, filed on October 21, 2020, incorporated herein by reference.

FIELD OF THE INVENTION

The present invention generally relates to the field of genetic engineering and antibody-based medicine. In particular, the present invention relates to antibodies directed against tetanus toxin and their use.

BACKGROUND ART

Tetanus is an acute and fatal disease caused by neurotoxins secreted by Clostridium tetani and affects humans and animals. The most susceptible animals are horses, mules or donkeys from the order Perssodactyla. Humans are also susceptible to neurotoxins secreted by Clostridium tetani. This disease can be detected at any age. Neonatal tetanus is one of the leading causes of neonatal mortality in economically underdeveloped areas. The incubation period for tetanus is usually 3-21 days, generally about 10 days, but it can also range from one day to several months depending on the characteristics, extent and location of the wound. Typical clinical manifestations of tetanus include clenching of the teeth, increased stress on the motor nerve center, and local or systemic muscular spasmolytic paralysis. Patients often die from asphyxia and systemic organ failure. Even in modern intensive care units, the mortality rate for this disease is very high. Mortality rates from tetanus can range from 19% to 31%, especially after major natural disasters. There are three clinical types of tetanus, namely systemic tetanus, local tetanus and capitate tetanus, which in all cases account for approximately 88%, approximately 12% and approximately 1% respectively.

Clostridium tetani is a strictly anaerobic gram-positive bacterium. It has a flagellum, but no capsule. Its spores are widely found in fertilized soil, street dust, rancid dirt, in the intestines of animals and contaminated surfaces of objects. Clostridium tetani enters the human body primarily through a wound. If the wound is narrow and deep, or if the wound is simultaneously infected with aerobic pyogenic bacteria, then Clostridium tetani will multiply in large numbers under anaerobic conditions and produce three exotoxins, among which tetanus toxin causes the characteristic symptoms of tetanus and leads to the production of protective antibodies [1]. However, approximately 20% of patients do not have obvious invasive wounds, suggesting that Clostridium tetani can also enter the body through small scratches. Clostridium tetani can be divided into ten serotypes based on flagellar antigen agglutination reactions, but all serotypes produce the same neurotoxin. Toxins produced by individual strain types can be neutralized by any type of antitoxin. Tetanus toxin is highly toxic, second only to botulinum toxin. Its lethal dose for mice is only 2-6 ng/kg.

Tetanus toxin, also known as tetanus neurotoxin, is a single-chain protein that has a relative molecular weight of approximately 150 kDa, consists of 1315 amino acid residues and can be cleaved into a light chain (fragment A, 50 kDa) and a heavy chain (HC, 100 kDa) , linked through a disulfide bond, thereby forming its active form [2]. Fragment A is a zinc metalloproteinase that, when released into the cytoplasm, cleaves vesicle-associated membrane protein-2 (VAMP-2) and blocks the release of inhibitory neurotransmitters such as glycine and glycine-aminobutyric acid (GABA), resulting in antispasmodic muscle paralysis. [3]. The heavy chain contains two functional domains. The C-terminal domain (fragment C) binds to the surfaces of nerve cells in such a way that the toxin molecule is endocytosed into vesicles. The N-terminal domain (fragment B) passes through the vesicle membrane and transports fragment A into the cytoplasm of the neuron [4]. Fragment C is divided into a C-terminal subdomain (CTS) and an N-terminal subdomain (HCN) [5]. The process by which tetanus toxin enters nerve cells is not fully understood. The dual receptor mechanism is now generally accepted and includes the ganglioside receptor, in particular GTlb and GDlb, and a protein receptor [6].

Tetanus is a preventable disease. Subjects may receive active or passive immunization treatments following injury. Humoral immunity has a protective effect against tetanus. Neutralizing antibodies bind to the toxin and prevent the toxin from interacting with receptors on target cells and subsequent internalization of the toxin into the cell. Clinically, passive immunization is recommended for injured patients who have an unclear immunization history or are not immunized, have not received a complete primary immunization, or have received a complete primary immunization but with the last booster immunization received more than 5 years ago. Currently, there are two types of passive immunization drugs for the prevention and treatment of tetanus in the clinic. One is tetanus antitoxin (TAT), which is obtained from equine plasma immunized with tetanus toxoid by enzymatic digestion and desalting. A skin test must be performed before using TAT. TAT often causes allergic reactions with an incidence of 5-30%. Sometimes anaphylactic shock occurs. Another type of passive immunization preparation is human tetanus immunoglobulin (HIGT), which is obtained by collecting plasma or serum with a high titer of tetanus antibody from blood donors sequentially immunized with hepatitis B vaccine and tetanus toxoid, and treating the plasma or serum with low temperature method with ethanol. HIGT can be injected directly without a skin test. However, since HIGT is a blood product, there is a potential risk of infectious diseases such as hepatitis C and AIDS. Additionally, HIGT is source limited and has low yield, high cost, and inconsistent quality between batches [7].

Antibody-based drugs can overcome some of the above-mentioned disadvantages and have certain advantages such as production through stable expression by mammalian cell-based expression systems and a defined composition.

Due to clinical needs, there is great medical importance in developing antibodies that can neutralize tetanus toxin and be used in the prevention and treatment of tetanus.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, there is provided a bispecific antibody comprising a first antigen binding fragment and a second antigen binding fragment that binds to different epitopes of tetanus toxin, wherein the bispecific antibody has tetanus toxin neutralizing activity.

In some embodiments of the first aspect, said first antigen binding fragment comprises HCDR1 having the amino acid sequence SYWIY (SEQ ID NO: 1), HCDR2 having the amino acid sequence EINPTNGFANYNEKFKT (SEQ ID NO: 2), or EINPTAGFANYNEKFKT (SEQ ID NO: 3), or EINPTNAFANYNEKFKT (SEQ ID NO: 4), HCDR3 having the amino acid sequence HFRFPY (SEQ ID NO: 5), LCDR1 having the amino acid sequence RASQDIGSSLT (SEQ ID NO: 6), LCDR2 having the amino acid sequence ATSSLDS (SEQ ID NO: 7), and LCDR3 having the amino acid sequence LQYASSPYT (SEQ ID NO: 8); wherein the amino acid sequences HCDR and LCDR are defined according to Kabat.

In some embodiments of the first aspect, said second antigen binding fragment comprises HCDR1 having the amino acid sequence DYGVN (SEQ ID NO: 9), HCDR2 having the amino acid sequence MIWSDGTTDYSSALKS (SEQ ID NO: 10), HCDR3 having the amino acid sequence VDGYSHYYAMDY (SEQ ID NO: 11 ), LCDR1 having the amino acid sequence RASENIYSYLA (SEQ ID NO: 12), LCDR2 having the amino acid sequence NAKTLAE (SEQ ID NO: 13), and LCDR3 having the amino acid sequence QHHYGLPFT (SEQ ID NO: 14); wherein the amino acid sequences HCDR and LCDR are defined according to Kabat.

In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the first antigen binding fragment is as set forth in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region of the first antigen binding fragment is as set forth in SEQ ID NO: 18; or the amino acid sequence of the heavy chain variable region of the first antigen binding fragment is as set forth in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region of the first antigen binding fragment is as set forth in SEQ ID NO: 18; or the amino acid sequence of the heavy chain variable region of the first antigen binding fragment is as set forth in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region of the first antigen binding fragment is as set forth in SEQ ID NO: 18.

In some embodiments of the first aspect, the amino acid sequence of the second antigen binding fragment heavy chain variable region is as set forth in SEQ ID NO: 19, and the amino acid sequence of the second antigen binding fragment light chain variable region is as set forth in SEQ ID NO: 20.

In some embodiments of the first aspect, the forms of the first antigen binding fragment and the second antigen binding fragment are independently selected from a single chain variable fragment (scFv) or a Fab fragment.

A second aspect of the invention provides a monoclonal antibody that binds to tetanus toxin, comprising:

HCDR1 having the amino acid sequence SYWIY (SEQ ID NO: 1), HCDR2 having the amino acid sequence EINPTNGFANYNEKFKT (SEQ ID NO: 2), or EINPTAGFANYNEKFKT (SEQ ID NO: 3), or EINPTNAFANYNEKFKT (SEQ ID NO: 4), HCDR3, having the amino acid sequence HFRFPY (SEQ ID NO: 5), LCDR1 having the amino acid sequence RASQDIGSSLT (SEQ ID NO: 6), LCDR2 having the amino acid sequence ATSSLDS (SEQ ID NO: 7), and LCDR3 having the amino acid sequence LQYASSPYT (SEQ ID NO: 8); wherein the amino acid sequences HCDR and LCDR are defined according to Kabat.

The third aspect of the invention provides a monoclonal antibody that binds to tetanus toxin, comprising:

HCDR1 having the amino acid sequence DYGVN (SEQ ID NO: 9), HCDR2 having the amino acid sequence MIWSDGTTDYSSALKS (SEQ ID NO: 10), HCDR3 having the amino acid sequence VDGYSHYYAMDY (SEQ ID NO: 11), LCDR1 having the amino acid sequence RASENIYSYLA (SEQ ID NO: 12), LCDR2 having the amino acid sequence NAKTLAE (SEQ ID NO: 13), and LCDR3 having the amino acid sequence QHHYGLPFT (SEQ ID NO: 14); wherein the amino acid sequences HCDR and LCDR are defined according to Kabat.

A fourth aspect of the invention provides a pharmaceutical composition comprising a bispecific antibody according to the first aspect or a monoclonal antibody according to the second or third aspect and a pharmaceutically acceptable excipient, diluent or carrier.

A fifth aspect of the invention provides the use of a bispecific antibody according to the first aspect, a monoclonal antibody according to the second or third aspect, or a pharmaceutical composition according to the fourth aspect in the manufacture of a medicament for the prevention or treatment of tetanus.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1 shows the results of an ELISA (enzyme-linked immunosorbent assay) assay, including an assessment of how the anti-TT-Hc monoclonal antibodies S22E2-mIgG2a and S24B8-mIgG2a inhibit the binding of purified phage S22E2 to TT-Hc.

Fig. 2 shows the results of an ELISA assay including an assessment of how the anti-TT-Hc monoclonal antibodies S22E2-mIgG2a and S24B8-mIgG2a inhibit the binding of purified phage S24B8 to TT-Hc.

Fig. 3 shows the results of an ELISA assay including an assessment of how the anti-TT-Hc monoclonal antibodies S22E2-mIgG2a and S24B8-mIgG2a inhibit the binding of TT-Hc to the ganglioside GTlb.

Fig. 4 demonstrates the neutralizing effects of anti-tetanus toxin monoclonal antibodies against tetanus toxin.

Fig. 5 demonstrates the protective effect of a combination of anti-tetanus toxin monoclonal antibodies against lethal tetanus toxin challenge in Balb/c mice.

Fig. 6 demonstrates that the bispecific antibody S22E2hl-scFv-FcHl+S24B8h3-IgG1K inhibits the binding of TT-Hc to GM1 ganglioside.

Fig. 7 demonstrates that the bispecific antibody S22E2hl-scFv-FcHl+S24B8h3-IgG1K inhibits the binding of TT-Hc to ganglioside GD3.

Fig. 8 demonstrates that the bispecific antibody S22E2hl-scFv-FcHl+S24B8h3-IgG1K inhibits the binding of TT-Hc to ganglioside GT1b.

Fig. 9 demonstrates the neutralizing activity of the anti-tetanus toxin bispecific antibody S22E2h1-scFv-FcH1+S24B8h3-IgG1K.

DESCRIPTION OF SEQUENCES

SEQ ID NO: 1 shows the amino acid sequence of the HCDR1 humanized heavy chain variable region mutants S24B8VH-h1, S24B8VH-h2 and S24B8VH-h3.

SEQ ID NO: 2 shows the amino acid sequence HCDR2 of the humanized heavy chain variable region mutant S24B8VH-h1.

SEQ ID NO: 3 shows the HCDR2 amino acid sequence of the humanized heavy chain variable region mutant S24B8VH-h2.

SEQ ID NO: 4 shows the HCDR2 amino acid sequence of the humanized heavy chain variable region mutant S24B8VH-h3.

SEQ ID NO: 5 shows the HCDR3 amino acid sequence of the humanized heavy chain variable region mutants S24B8VH-h1, S24B8VH-h2 and S24B8VH-h3.

SEQ ID NOs: 6-8 show, respectively, the amino acid sequences LCDR1, LCDR2 and LCDR3 of the humanized light chain variable region mutant S24B8VK-h1.

SEQ ID NOs: 9-11 show, respectively, the amino acid sequences HCDR1, HCDR2 and HCDR3 of the humanized heavy chain variable region mutant S22E2VH-h1.

SEQ ID NOs: 12-14 show, respectively, the amino acid sequences LCDR1, LCDR2 and LCDR3 of the humanized light chain variable region mutant S22E2VK-h1.

SEQ ID NO: 15 shows the amino acid sequence of the humanized heavy chain variable region mutant S24B8VH-h1.

SEQ ID NO: 16 shows the amino acid sequence of the humanized heavy chain variable region mutant S24B8VH-h2.

SEQ ID NO: 17 shows the amino acid sequence of the humanized heavy chain variable region mutant S24B8VH-h3.

SEQ ID NO: 18 shows the amino acid sequence of the humanized light chain variable region mutant S24B8VK-h1.

SEQ ID NO: 19 shows the amino acid sequence of the humanized heavy chain variable region mutant S22E2VH-h1.

SEQ ID NO: 20 shows the amino acid sequence of the humanized light chain variable region mutant S22E2VK-h1.

SEQ ID NO: 21 shows the amino acid sequence S24B8VH-h1+CH-IgG1K.

SEQ ID NO: 22 shows the amino acid sequence S24B8VH-h2+CH-IgG1K.

SEQ ID NO: 23 shows the amino acid sequence S24B8VH-h3+CH-IgG1K.

SEQ ID NO: 24 shows the amino acid sequence of S24B8VK-h1+CK.

SEQ ID NO: 25 shows the amino acid sequence of S22E2-h1-scFv-FcHl.

SEQ ID NO: 26 shows the amino acid sequence of the recombinant tetanus toxin heavy chain C-terminal domain (TT-Hc) protein.

SEQ ID NO: 27 shows the amino acid sequence of the His terminal sequence.

SEQ ID NO: 28 shows the amino acid sequence of the heavy chain constant region of the human (homo sapiens) IgG1 subtype.

SEQ ID NO: 29 shows the amino acid sequence of the heavy chain constant region of the murine (mus musculus) IgG2a subtype.

SEQ ID NO: 30 shows the amino acid sequence of the IgG1H mutant of the human IgG1 subtype.

SEQ ID NO: 31 shows the amino acid sequence of the IgG1K mutant of the human IgG1 subtype.

SEQ ID NO: 32 shows the amino acid sequence of the murine IgG2a subtype mIgG2a-H mutant.

SEQ ID NO: 33 shows the amino acid sequence of the murine IgG2a subtype mIgG2aK mutant.

SEQ ID NO: 34 shows the amino acid sequence of the human (homo sapiens) kappa subtype light chain constant region.

SEQ ID NO: 35 shows the amino acid sequence of the human (homo sapiens) lambda light chain constant region.

SEQ ID NO: 36 shows the amino acid sequence of the murine (mus musculus) limbda subtype light chain constant region.

SEQ ID NO: 37 shows the amino acid sequence of the murine (mus musculus) lambda subtype light chain constant region.

SEQ ID NO: 38 shows the nucleotide sequence of the PmCGR primer.

SEQ ID NO: 39 shows the nucleotide sequence of the PmCKR primer.

SEQ ID NO: 40 shows the amino acid sequence of the Fc segment containing the Knob mutation (FcK).

SEQ ID NO: 41 shows the amino acid sequence of the Fc segment containing the Hole mutation (FcHl).

DETAILED DESCRIPTION OF THE INVENTION

The inventors of the present invention have conducted extensive research and development of anti-tetanus antibody drugs and have produced novel bispecific antibodies and monoclonal antibodies against tetanus toxin using antibody engineering technology. Tetanus toxin is a macromolecular protein that consists of several domains. Theoretically, it contains multiple epitopes that can induce humoral immunity, and antibodies that bind to different epitopes can be obtained from immunized subjects. The C fragment of tetanus toxin is a receptor-binding domain, and it is likely that antibodies against the C fragment have neutralizing activities by preventing the toxin from binding to the gangliosides GT1b, GD1b and GM1, and achieving neutralizing effects on the toxin.

Various aspects of the present invention provide novel bispecific antibodies and monoclonal antibodies against tetanus toxin, polynucleotides encoding such bispecific antibodies or monoclonal antibodies, vectors containing such polynucleotides, host cells containing such polynucleotides or vectors, methods for producing and purifying such bispecific antibodies, or monoclonal antibodies and the medical and biological applications of such bispecific antibodies or monoclonal antibodies. Based on the variable region sequences of bispecific antibodies or monoclonal antibodies provided herein, full-length bispecific antibody molecules or monoclonal antibody molecules can be designed for clinical use as a drug for the prevention or treatment of tetanus.

Unless otherwise indicated, the invention according to this application can be practiced using conventional molecular biology, microbiology, cell biology, biochemistry and immunological methods in the art.

Unless otherwise specified, terms used in this specification have the meanings commonly understood by one skilled in the art.

DEFINITIONS

As used herein, the term “antibody” refers to an immunoglobulin molecule that is capable of specifically binding to a target through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, and polypeptides. As used herein, "antibody" includes not only the intact (i.e., full-length) antibody, but also an antigen-binding fragment thereof (eg, Fab, Fab', F(ab') ₂ , Fv), a variant thereof, a fusion protein containing fragments antibodies, humanized antibody, chimeric antibody, diabody, linear antibody, single chain antibody, multispecific antibody (eg, bispecific antibody), and any other modified immunoglobulin molecule formats containing the desired specific antigen recognition site, including a glycosylated variant of the antibody, a variant of the amino acid sequence, and a covalently modified antibody .

Typically, an intact or full-length antibody contains two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and first, second and third constant regions (CH1, CH2 and CH3). Each light chain contains a variable region (VL) and a light chain constant region (CL). A full-length antibody can be any type of antibody, such as an IgD, IgE, IgG, IgA, or IgM antibody (or subtypes thereof), but is not necessarily of any particular type. Immunoglobulins can be classified into different types depending on their heavy chain constant domain amino acid sequences. Generally, there are five main types of immunoglobulins, i.e. IgA, IgD, IgE, IgG and IgM, and some of these types can be further classified into subtypes (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains corresponding to the individual immunoglobulin types are referred to as αδ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of various types of immunoglobulins are well known.

As used herein, the term "bispecific antibody" is an antibody that has the ability to bind to two different antigens. The bispecific antibody may have various structural configurations. For example, a bispecific antibody may consist of two Fc fragments and two antigen binding fragments fused to them, respectively (similar to a natural antibody, except that the two arms bind to different antigenic targets or epitopes). Antigen binding fragments may be in the form of scfv or Fab fragments. When two antigens are given, then each of the two different antigen-binding fragments of the bispecific antibody binds to the N-terminus of one Fc fragment. The configuration of the antigen-binding fragments of the two arms can have four combinations, i.e. scfv+Fab fragment, Fab+scfv fragment, scfv+scfv and Fab fragment+Fab fragment. Fc fragments may contain mutations that can cause heavy chain heteromerization. The KIH (knob-in-hollow) method is one of the strategies related to heavy chain heteromerization. Generally, the KIH method refers to the formation of a structure that facilitates the pairing of heterogeneous half-bodies by engineering the amino acid sequence of the CH3 region. The latter makes it possible to maximally maintain the structure of a normal antibody during the formation of a bispecific antibody. For guidance on the KIH method, see, for example, "An efficient route to human bispecific IgG", A. Margaret Merchant et al., Nature Biotechnology, Volume 16, 1998, which is incorporated herein by reference in its entirety. In addition, the bispecific antibody may be configured such that an antibody that binds to a first antigen (e.g., in the form of a natural antibody) has an antigen binding moiety that can bind to a second antigen extending from the C terminus of the CH3 region (e.g., via a flexible linker).

As used herein, the terms "antigen-binding portion" or "antigen-binding fragment" may be used interchangeably, and refer to the portion or region of an intact antibody molecule responsible for binding to an antigen. The antigen binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of VH and VL typically contains three regions that determine complementarity, i.e. CDR1, CDR2 and CDR3.

It is well known to one skilled in the art that complementarity determining regions (CDRs, typically including CDR1 CDR2 and CDR3) are portions of the variable region that primarily affect the affinity and specificity of an antibody. VH or VL CDR sequences have two general definitions, that is, the Kabat definition and the Chothia definition (see, for example, Kabat, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al, J Mol Biol 273: 927-948 (1997); and Martin et al, Proc Natl Acad Sci USA 86: 9268-9272 (1989). For the variable region sequences of a given antibody, the sequences of the CDR regions in VH and VL can be determined in accordance with the Kabat definition or the Chothia definition. In an embodiment of the present invention, the CDR sequences are determined according to Kabat.

For the variable region sequences of a given antibody, the sequences of the CDR regions in the variable region sequences can be analyzed in various ways, for example, using the online software Abysis (http://www.abysis.org/).

For conventional antibodies, examples of the antigen binding fragment include, but are not limited to (1) a Fab fragment, which may be a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) an F(ab') ₂ moiety, which may be a divalent moiety having two Fab' moieties linked by a hinge region disulfide bridge (ie, a Fab'dimer); (3) an Fv fragment having VL and VH domains in one arm of the antibody; (4) single chain Fv (scFv), which may be a single polypeptide chain consisting of a VH domain and a VL domain linked by a polypeptide linker; and (5) (scFv) ₂ , which may contain two VH domains linked by a peptide linker, and two VL domains that are combined with two VH domains via a disulfide bridge.

When constructing a bispecific antibody, an "antigen-binding fragment" includes, without limitation, a Fab fragment or a single chain variable fragment (scFv).

As used herein, the term “single chain variable fragment (scFv)” refers to an antibody with a single chain structure, typically engineered using genetic engineering techniques, comprising a polypeptide chain comprising a heavy chain variable region (VH) and a light chain variable region (VL). A flexible linker is typically designed between the heavy chain variable region and the light chain variable region so that the heavy chain variable region and the light chain variable region can fold into the correct conformation for binding to the antigen.

As used herein, the term "Fab (antigen-binding) fragment", "Fab fragment" and the like. refers to an antibody fragment that is obtained after treating an intact antibody with papain and that is capable of binding to an antigen including an intact light chain (VL-CL), a heavy chain variable region, and a CHI fragment (VH-CH1).

As used herein, the term “monoclonal antibody” refers to an antibody from a substantially homogeneous population of antibodies, meaning that the antibodies comprising the population are the same except for naturally occurring mutations that may occur in a small number of individual antibodies. Monoclonal antibodies described herein particularly include “chimeric” antibodies in which a fragment of the heavy and/or light chain is identical or homologous to the corresponding sequence in the antibody derived from a particular species or belonging to a particular type or subtype of the antibody, while the remainder of the heavy and/or light chain /or the light chain is identical or homologous to the corresponding sequence in the antibody, derived from other species or belonging to another type or subtype of antibody, and also includes fragments of such antibodies, provided that they demonstrate the desired biological activity (see US patent No. 4816567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)).

As used herein, the term "specific binding" refers to an involuntary binding reaction between two molecules, for example, the binding of an antibody to an epitope of an antigen.

In a first aspect of the present invention, there is provided a bispecific antibody comprising a first antigen binding fragment and a second antigen binding fragment that binds to different epitopes of tetanus toxin, wherein the bispecific antibody has tetanus toxin neutralizing activity.

In some embodiments of the first aspect, said first antigen binding fragment comprises HCDR1 having the amino acid sequence SYWIY (SEQ ID NO: 1), HCDR2 having the amino acid sequence EINPTNGFANYNEKFKT (SEQ ID NO: 2), or EINPTAGFANYNEKFKT (SEQ ID NO: 3), or EINPTNAFANYNEKFKT (SEQ ID NO: 4), HCDR3 having the amino acid sequence HFRFPY (SEQ ID NO: 5), LCDR1 having the amino acid sequence RASQDIGSSLT (SEQ ID NO: 6), LCDR2 having the amino acid sequence ATSSLDS (SEQ ID NO: 7), and LCDR3 having the amino acid sequence LQYASSPYT (SEQ ID NO: 8); wherein the amino acid sequences HCDR and LCDR are defined according to Kabat.

In some embodiments of the first aspect, said second antigen binding fragment comprises HCDR1 having the amino acid sequence DYGVN (SEQ ID NO: 9), HCDR2 having the amino acid sequence MIWSDGTTDYSSALKS (SEQ ID NO: 10), HCDR3 having the amino acid sequence VDGYSHYYAMDY (SEQ ID NO: 11 ), LCDR1 having the amino acid sequence RASENIYSYLA (SEQ ID NO: 12), LCDR2 having the amino acid sequence NAKTLAE (SEQ ID NO: 13), and LCDR3 having the amino acid sequence QHHYGLPFT (SEQ ID NO: 14); wherein the amino acid sequences HCDR and LCDR are defined according to Kabat.

In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the first antigen binding fragment is as set forth in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 18; or the amino acid sequence of the heavy chain variable region of the first antigen binding fragment is as set forth in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region of the first antigen binding fragment is as set forth in SEQ ID NO: 18; or the amino acid sequence of the heavy chain variable region of the first antigen binding fragment is as set forth in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region of the first antigen binding fragment is as set forth in SEQ ID NO: 18.

In some embodiments of the first aspect, the amino acid sequence of the second antigen binding fragment heavy chain variable region is as set forth in SEQ ID NO: 19, and the amino acid sequence of the second antigen binding fragment light chain variable region is as set forth in SEQ ID NO: 20.

In some embodiments of the first aspect, the forms of the first antigen binding fragment and the second antigen binding fragment are independently selected from a single chain variable fragment (scFv) or a Fab fragment.

In some embodiments of the first aspect, said first antigen binding fragment is a Fab fragment and said second antigen binding fragment is a single chain variable fragment (scFv).

In some embodiments of the first aspect, the bispecific antibody comprises the amino acid sequence set forth in SEQ ID NOs: 21, 22, and 23 and the amino acid sequence set forth in SEQ ID NO: 24.

In some embodiments of the first aspect, the bispecific antibody comprises the amino acid sequence set forth in SEQ ID NO: 25.

A second aspect of the invention provides a monoclonal antibody that binds to tetanus toxin, comprising:

HCDR1 having the amino acid sequence SYWIY (SEQ ID NO: 1), HCDR2 having the amino acid sequence EINPTNGFANYNEKFKT (SEQ ID NO: 2), or EINPTAGFANYNEKFKT (SEQ ID NO: 3), or EINPTNAFANYNEKFKT (SEQ ID NO: 4), HCDR3, having the amino acid sequence HFRFPY (SEQ ID NO: 5), LCDR1 having the amino acid sequence RASQDIGSSLT (SEQ ID NO: 6), LCDR2 having the amino acid sequence ATSSLDS (SEQ ID NO: 7), and LCDR3 having the amino acid sequence LQYASSPYT (SEQ ID NO: 8); wherein the amino acid sequences HCDR and LCDR are defined according to Kabat.

In some embodiments of the second aspect, the monoclonal antibody comprises the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 15 and the light chain variable region amino acid sequence set forth in SEQ ID NO: 18; or the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 16 and the light chain variable region amino acid sequence set forth in SEQ ID NO: 18; or the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 17 and the light chain variable region amino acid sequence set forth in SEQ ID NO: 18.

The third aspect of the invention provides a monoclonal antibody that binds to tetanus toxin, comprising:

HCDR1 having the amino acid sequence DYGVN (SEQ ID NO: 9), HCDR2 having the amino acid sequence MIWSDGTTDYSSALKS (SEQ ID NO: 10), HCDR3 having the amino acid sequence VDGYSHYYAMDY (SEQ ID NO: 11), LCDR1 having the amino acid sequence RASENIYSYLA (SEQ ID NO: 12), LCDR2 having the amino acid sequence NAKTLAE (SEQ ID NO: 13), and LCDR3 having the amino acid sequence QHHYGLPFT (SEQ ID NO: 14); wherein the amino acid sequences HCDR and LCDR are defined according to Kabat.

In some embodiments of the third aspect, the monoclonal antibody comprises the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 19 and the light chain variable region amino acid sequence set forth in SEQ ID NO: 20.

A fourth aspect of the invention provides a pharmaceutical composition comprising a bispecific antibody according to the first aspect or a monoclonal antibody according to the second or third aspect and a pharmaceutically acceptable excipient, diluent or carrier.

In some embodiments of the fourth aspect, the pharmaceutical composition is for the prevention or treatment of tetanus.

In some embodiments of the fourth aspect, the pharmaceutical composition may further comprise one or more of a lubricant such as talc, magnesium stearate and mineral oil; wetting agent; emulsifier; suspending agent; a preservative such as benzoic acid, sorbic acid and calcium propionate; sweetener and/or flavoring agent.

In some embodiments of the fourth aspect, the pharmaceutical composition herein may be formulated as a tablet, pill, powder, lozenge, elixir, suspension, emulsion, solution, syrup, suppository, or capsule.

In some embodiments of the fourth aspect, the pharmaceutical composition in accordance with the present invention can be delivered using any physiologically acceptable route of administration, including, without limitation, oral administration, parenteral administration, nasal administration, rectal administration, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration or inhalation administration.

In some embodiments of the fourth aspect, a pharmaceutical composition for therapeutic use can be prepared for storage in a lyophilized composition or in the form of an aqueous solution by admixing the agent having the desired purity with a pharmaceutically acceptable carrier or excipient, as appropriate.

A fifth aspect of the invention provides the use of a bispecific antibody according to the first aspect, a monoclonal antibody according to the second or third aspect, or a pharmaceutical composition according to the fourth aspect in the manufacture of a medicament for the prevention or treatment of tetanus.

A sixth aspect of the invention provides a method for preventing or treating tetanus, comprising administering to a subject in need thereof a bispecific antibody according to the first aspect, a monoclonal antibody according to the second or third aspect, or a pharmaceutical composition according to the fourth aspect.

In other aspects of the invention, there is provided a nucleic acid molecule encoding a bispecific antibody in accordance with the first aspect or a monoclonal antibody in accordance with the second or third aspect. In some embodiments, the nucleic acid molecule is operably linked to a regulatory sequence that can be recognized by a host cell transformed by the vector.

The invention also provides a vector containing an isolated nucleic acid molecule encoding a bispecific antibody in accordance with the first aspect or a monoclonal antibody in accordance with the second or third aspect, and a host cell containing the nucleic acid molecule or vector.

In other aspects of the invention, a method for producing a bispecific antibody in accordance with the first aspect or a monoclonal antibody in accordance with the second or third aspect is provided. In some embodiments, a method of producing a bispecific antibody in accordance with the first aspect or a monoclonal antibody in accordance with the second or third aspect includes culturing the host cell in such a way as to facilitate expression of the nucleic acid. In some embodiments, a method for producing a bispecific antibody in accordance with the first aspect or a monoclonal antibody in accordance with the second or third aspect further comprises isolating the bispecific antibody or monoclonal antibody from a host cell culture medium.

It should be understood that the foregoing detailed description is intended only to enable one skilled in the art to better understand the present invention and is not intended to be limiting in any way. Various modifications and variations may be made to the described embodiments by one skilled in the art.

The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

Examples

Example 1: Preparation of recombinant tetanus toxin antigen and recombinant antibodies

1.1 Preparation of recombinant tetanus toxin antigen Recombinant tetanus toxin heavy chain C-terminal domain protein (TT-Hc, SEQ ID NO: 26) was used in the production of monoclonal antibodies against tetanus toxin. This protein was derived from Clostridium tetani and lacks any post-translational modification and can therefore be expressed using the E. coli expression system. In addition, a His terminal sequence (His, SEQ ID NO: 27) was added to the C-terminus of the recombinant protein to facilitate purification of the recombinant protein and identification of monoclonal antibody function.

The gene encoding TT-Hc (containing the His terminal sequence) was designed and synthesized according to the amino acid sequences of recombinant TT-Hc proteins in the Uniprot database. The synthesized gene was cloned into a suitable eukaryotic expression vector (such as pET-22b, mvitrogen, Inc.) using conventional molecular biology techniques. Expression plasmids were transformed into competent E. coli cells (such as BL21 (DE3), Solarbio, Inc.). E. coli expressing recombinant TT-Hc-His protein was induced to express with IPTG (isopropyl-beta-0-1-thiogalactopyranoside) (such as 18070-lg, Solarbio, Inc.). The bacteria were then collected by centrifugation and disrupted using an ultrasonic cell disruptor (such as VCX130, Sonics, Inc.). Supernatants were collected by centrifugation. The recombinant protein in the supernatants was further purified using a metal chelated affinity chromatography column (such as HisTrap FF, GE, Inc.). The recombinant protein preservation buffer was then changed to PBS (phosphate buffered saline) (pH 7.0) or other suitable buffers using a desalting column (such as Hitrap desaulting, GE, Inc.). If necessary, the sample can be sterilized by filtration and then stored in aliquots at -20°C.

1.2. Preparation of recombinant antibodies

Nucleotide sequences encoding the heavy chain variable region and the variable light chain region of the antibody were cloned into eukaryotic expression vectors (such as pcDNA3.1, Invitrogen, Inc.) carrying, respectively, nucleotide sequences encoding the heavy chain constant region and the light chain constant region, using conventional molecular biological methods. Intact antibodies were expressed in combination. The antibody heavy chain constant regions may be of the human IgG1 (SEQ ID NO: 28) or murine IgG2a (SEQ ID NO: 29) subtype, or may be an IgG1H mutant of the human IgG1 (SEQ ID NO: 30) and IgG1K (SEQ ID NO: 31) based on KIH (ridge-in-hole) technology, or may be a mIgG2a-H mutant of the murine IgG2a subtype (SEQ ID NO: 32) and mIgG2aK (SEQ ID NO: 33). The light chain constant regions may be human kappa subtype (SEQ ID NO: 34), human lambda subtype (SEQ ID NO: 35), mouse kappa subtype (SEQ ID NO: 36), or mouse lambda subtype (SEQ ID NO: 37).

The resulting recombinant antibody expression plasmids were transfected into HEK293 cells (such as HEK293F, Invitrogen, Inc.) using liposomes (such as 293fectin, Invitrogen, Inc.) or other transfection reagents (such as PEI). Cells were cultured in suspension in serum-free media for 3-5 days. Culture supernatants were then collected by centrifugation. The recombinant protein in the supernatant was further purified using a ProteinA/G affinity chromatography column (such as Mabselect SURE, GE, Inc.). The recombinant protein preservation buffer was then replaced with PBS (pH 7.0) or other suitable buffers using a desalting column (such as Hitrap desaulting, GE, Inc.). If necessary, the antibody sample can be sterilized by filtration and then stored in aliquots at -20°C.

Example 2: Immunization of mice and construction of immune library BALB/c mice aged 6 to 8 weeks were immunized with the recombinant TT-Hc-His protein obtained in Example 1 as antigen at an immunization dose of 50 μg per mouse. Booster immunization was carried out every 14 days. Mice were sacrificed 8 weeks after primary immunization and spleen cells were collected. Mouse spleen lymphocytes were isolated using Mouse Lymphocyte Isolation Solution (Dakewe Biotech Co., Ltd., CAT #DKW33-R0100). Total RNA was extracted from isolated lymphocytes using a total RNA cell extraction kit (Tiangen Biotech (Beijing) Co., Ltd., CAT #DP430). Using the isolated total RNA as a template, the antibody heavy chain variable region and light chain variable region were synthesized using a first-strand cDNA synthesis kit (Thermo scientific, CAT#K1621). The reverse transcription primers used were gene specific primers, and the primer pairing regions were located in the heavy chain constant region and the light chain constant region of the antibody, respectively. The specific sequences were, respectively, PmCGR: TGCATTTGAACTCCTGCC (SEQ ID NO: 38), and PmCKR: CCATCAATCTCCACTGAC (SEQ ID NO: 39). The synthesized cDNA was immediately stored at -70°C for later use. Then, using the cDNA obtained by reverse transcription as a template, primers were synthesized according to Krebber A, Bornhauser S, Burmester J, et al. Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J Immunol Methods. 1997; 201(1):35-55, the entire contents of which are incorporated herein by reference, and the nucleotide sequences encoding the mouse antibody VH and VK were respectively amplified by PCR. Single chain variable fragment (scFv) genes were then constructed using overlap extension PCR technology. Finally, the nucleotide sequences of the obtained mouse single-chain antibodies were cloned into the pADSCFV-S vector (see Example 1 in Chinese Patent Application No. 201510097117.0 for detailed experimental protocols, the contents of which are incorporated herein by reference) to construct a scFv library. The antibody library has a capacity of 5 × 10E8 and an accuracy of 50%.

Example 3: Screening and Preliminary Identification of a Mouse Immune Library

3.1. Mouse Immune Library Screening

Referring to Chinese Patent Application No. 201510097117.0 (the entire contents of which are incorporated herein by reference), the phage library exhibiting murine single chain antibodies constructed in Example 2 was screened using an enzyme-linked screening strategy (for the experimental protocol, see Phage Display: A Practical Approach, Tim Clackson and Henry B. Lowman (ed.), translated by Lan Ma et al., Chemical Industry Press, May 2008) using recombinant human TT-Hc-His prepared in Example 1 as antigen. Three rounds of screening were performed by binding, elution, neutralization, infection, and amplification. Finally, two single-chain antibodies S22E2 and S24B8 were obtained that specifically bind to human TT-Hc-His.

The nucleotide sequences encoding the heavy chain and light chains of S22E2 and S24B8, respectively, were cloned into eukaryotic expression vectors carrying nucleotide sequences encoding the murine mIgG2a heavy chain constant region and the mouse kappa light chain constant region by using conventional molecular biological techniques to produce recombinant mouse antibodies.

3.2. Affinity analysis of recombinant monoclonal antibodies against TT-Hc The affinities of antibodies against TT-Hc were determined by surface plasmon resonance using Biacore X100. Reagents and consumables such as Amino Coupling Kit (BR-1000-50), Mouse Antibody Immobilization Kit (BR-1008-38), CM5 Chip (BR100012) and 10xHBS-EP pH 7.4 (BR100669) were purchased from GE Healthcare. The carboxylated CM5 chip surface was activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the kit instructions. Anti-mouse IgG (Fc) antibody (immobilized antibody) was diluted to 25 μg/ml using 10 mM sodium acetate (pH 5.0) and then injected at a flow rate of 10 μl/min in order to achieve a binding value of up to approximately 10,000 response units (RU). After injection of the immobilized antibody, 1 M ethanolamine was injected to block unreacted groups. To measure kinetics, anti-TT-Hc antibody was diluted to 0.5-1 μg/ml and injected at 10 μl/min to ensure that approximately 50 RU of antibody was captured by anti-TT-Hc antibody. mouse Fc. TT-Hc-His was then subjected to a series of concentration gradients (e.g., 6.17 nm, 18.5 nm, 55.6 nm, 167 nm, and 500 nm) and injected at 25°C from lower to higher concentrations at 30 μl /min with an association time of 120 s and a dissociation time of 3600 s. A 10 mM glycine-HCl solution (pH 2.0) was injected at 10 μL/min for 30 s to regenerate the chip surface. The rate of association (Ka) and rate of dissociation (Kd) were calculated by fitting the binding and dissociation sensorgrams to a 1:1 binding model using version 2.0.1 of the Biacore X100 evaluation software. The dissociation equilibrium constant (KD) was calculated as the ratio Kd/Ka. The fitting results are presented in Table 1.

Example 4: Anti-tetanus toxin monoclonal antibody epitope analysis 96-well ELISA plates were coated with recombinant TT-Hc-His protein (3 μg/ml, 100 μl/well) overnight in the refrigerator at 4°C and then blocked with blocking solution ( PBS-0.1% Tween20-3% milk) at 37°C for 1 hour. Mouse antibodies S22E2-mIgG2a and S24B8-mIgG2a were gradiently diluted with purified phage S22E2 (phage S22E2) at a fixed concentration (1 * 10 ^p CFU/ml). The initial antibody concentration was 50 μg/ml, and 3-fold dilution was performed for a total of 11 concentration gradients. Antibody dilutions were added to blocked 96-well ELISA plates at 100 μl/well and the plates were incubated at 37°C for 1 hour. The ELISA plates were then washed with PBS-0.1% Tween20, and then the secondary antibody anti-M13 with horseradish peroxidase (HRP) (Sino Biological Inc., 11973-MM05T-N) was added and the plates were incubated at 37°C for 1 hour . ELISA plates were washed with PBS-0.1% Tween20 and OPD substrate staining solution was added. After incubation for 5-10 minutes, staining was stopped with 1 M H2SO4. Optical density values were determined using a microplate reader at dual wavelength 492 nm/630 nm. The results of the ELISA assay (Fig. 1) demonstrated that S22E2-mIgG2a completely inhibited the binding signal between phage S22E2 and recombinant TT-Hc protein, but S24B8-mIgG2a had no effect on the binding of phage S22E2 to TT-Hc.

Similarly, S22E2-mIgG2a and S24B8-mIgG2a were subjected to gradient dilution with purified phage S24B8 (phage S24B8) at a fixed concentration. The binding signal between phage S24B8 and TT-Hc was detected using a secondary antibody anti-M13 with HRP. The ELISA results (Figure 2) demonstrated that S24B8-mIgG2a completely inhibited the binding signal between phage S24B8 and recombinant TT-Hc protein, but S22E2-mIgG2a had minimal effect on the binding of phage S24B8 to TT-Hc.

Thus, monoclonal antibodies against TT-Hc S22E2-mIgG2a and S24B8-mIgG2a bind to different epitopes of TT-Hc.

Example 5: Inhibition of TT-Hc binding to gangliosides using monoclonal antibodies against tetanus toxin

Tetanus toxin can bind to specific receptors on the surfaces of nerve cells through the Hc domain. The currently accepted mechanism is a dual receptor mechanism involving a ganglioside receptor and a protein receptor. 96-well ELISA plates were coated with GT1b gangliosides (100 μl/well), which were diluted to 10 μg/ml with methanol, and the plates were left overnight at room temperature to allow methanol to evaporate. The plates were then blocked with blocking solution (PBS-0.1% Tween20-1% BSA) at 37°C for 1 hour. TT-Hc was subjected to gradient dilution. The initial concentration of TT-Hc was 100 μg/ml, and 3-fold dilution was performed for a total of 7 concentration gradients. Mouse monoclonal antibodies against TT-Hc were diluted to 40 μg/ml using PBS and mixed with equal volumes of TT-Hc dilutions, and then the mixed solutions were added to blocked 96-well ELISA plates at 100 μl/well. A control group of TT-Hc without antibody was established. The plates were incubated at 37°C for 1 hour. ELISA plates were washed with PBS-0.1% Tween20 and anti-TT polyclonal antibody with HRP (prepared in the laboratory) was added at 100 μl/well. The plates were incubated at 37°C for 1 hour. The ELISA plates were then washed with PBS-0.1% Tween20 and OPD substrate staining solution was added. After incubation for 5-10 minutes, staining was stopped with 1 M H ₂ SO ₄ solution and absorbance values were determined using a microplate reader at dual wavelength 492 nm/630 nm. The results of the ELISA assay (Figure 3) showed that, relative to the binding curve of TT-Hc as a backbone, the binding curve shifted significantly to the right after the addition of S22E2-mIgG2a, indicating that S22E2-mIgG2a effectively inhibited the binding of TT-Hc to ganglioside GT1b. In contrast, S24B8-mIgG2a did not inhibit the binding of TT-Hc to ganglioside, and instead exhibited some stimulatory effects.

Example 6: Neutralizing activities of monoclonal antibodies against tetanus toxin

The neutralizing activity of the antibody was determined by mixing the antibody and a lethal dose of tetanus toxin in vitro and then injecting the mixture into Balb/c mice. Specifically, tetanus toxin was mixed with an anti-tetanus toxin monoclonal antibody or antitoxin, and the mixture was left at 37°C for 1 hour and then injected into the right hind leg muscle of Balb/c mice. The injected dose of tetanus toxin was 20×LD ₅₀ (median lethal dose)/mouse. The injected dose of commercially available equine tetanus immunoglobulin (F(ab') ₂ ) (labeled TAT, Shanghai Serum Bio-technology Co., LTD) was 1 IU/mouse. The injected dose of monoclonal antibody S22E2 or S24B8 was 10 μg/mouse. The dose of S24B8 and S22E2 in combination was 1.25 μg/mouse for each antibody. Balb/c mice injected with 20×LD ₅₀ tetanus toxin were used as a control group. There were a total of 5 groups, each containing 8 mice. Tetanus toxin was diluted to 800×LD ₅₀ /ml with boric acid buffer (ie 20×LD ₅₀ per 50 μl injection after mixing with antibody in equal volumes). Equine tetanus immunoglobulin was diluted to 40 IU/ml with boric acid buffer (i.e., 1 IU per 50 μl injection after mixing with equal volumes of tetanus toxin). S22E2 and S24B8 antibodies were diluted to 0.4 mg/ml (or 0.1 mg/ml) with boric acid buffer (i.e., for S22E2 or S24B8 treatment, 10 μg antibody per 50 μl injection after mixing with equal volumes of tetanus toxin ; and for treatment with S24B8 and S22E2 in combination, 1.25 μg of each antibody per 50 μl injection after mixing the mixture of the two antibodies (mixed in equal volumes) with tetanus toxin in equal volumes). Each animal was injected with 50 μl of a mixture of tetanus toxin and antibody. The experimental results are shown in Fig. 4. All Balb/c mice in the control group died of tetanus within 3 days after administration. Under experimental conditions, monoclonal antibody S24B8 or S22E2 partially neutralized the toxin and prolonged the survival of mice. In contrast, the combination of S24B8 and S22E2 completely neutralized the toxin, and all mice survived to the end of the experimental period, similar to the neutralizing activity found for equine tetanus immunoglobulin TAT.

Example 7: Anti-tetanus toxin monoclonal antibody combination protection in Balb/c mice lethally exposed to tetanus toxin

Balb/c mice were immunized before exposure to immunoglobulin, and antibody-treated mice were then given a lethal dose of tetanus toxin to determine the protective effects of the antibody. The injected dose of monoclonal antibodies S22E2 or S24B8 was 10 μg/mouse. The injection dose of S24B8 and S22E2 in combination was 1.25 μg/mouse for each antibody. Balb/c mice injected with 20×LD ₅₀ tetanus toxin were used as a control group. There were 4 groups in total, each of which included 8 mice.

Tetanus toxin was diluted to 400×LD ₅₀ /ml with boric acid buffer (ie containing 20×LD ₅₀ per 50 μl injection). Antibodies S22E2 and S24B8 were diluted to 0.2 mg/ml (or 0.05 mg/ml) with boric acid buffer (i.e., for treatment with S22E2 or S24B8, 10 μg of antibody per 50 μl injection; and for treatment with S24B8 and S22E2 in combinations of 1.25 μg of each antibody per 50 μl injection after mixing the two antibodies in equal volumes). 50 μl of anti-tetanus toxin antibody was injected into Balb/c mice, and 24 hours later the mice were injected with tetanus toxin into the muscles of the right hind legs. Each animal was injected with 50 μl of tetanus toxin. The experimental results are shown in Fig. 5. All Balb/c mice in the control group died of tetanus within 4 days after administration. Under these experimental conditions, the combination of S24B8 and S22E2 completely protected Balb/c mice, all of which survived at the end of the experimental period.

Example 8: Humanization and Deamination Site Mutation of Tetanus Toxin Monoclonal Antibodies

8.1. Humanization of Mouse Monoclonal Antibody S24B8 Mouse monoclonal antibody S24B8 was humanized to reduce its immunogenicity. The humanization scheme was adapted to the classical framework transplantation strategy (see Tan P, Mitchell DA, Buss TN, Holmes MA, Anasetti C, Foote J. "Superhumanized" antibodies: reduction of immunogenic potential by complementarity-determining region grafting with human germline sequences: application to an anti-CD28 J Immunol 2002;169(2):1119-1125, the entire contents of which are incorporated herein by reference). The genes encoding the variable regions of the heavy and light chains of S24B8 were respectively compared with the terminal genetic sequences of the human antibody in the IMGT database. Suitable terminal genetic sequences were selected to obtain the framework region 1-3 of the antibody (FR1+FR2+FR3), and a suitable genetic sequence of the J region was selected to obtain framework region 4 (FR4). This template can be selected depending on a variety of factors, such as the relative overall length of the antibody, the sizes of the CDRs, the amino acid residues at the junction of the framework region (FR) and the hypervariable region (CDR) of the antibody, and overall sequence homology. The selected template may be a mixture of multiple sequences or a common template to maximize the ability to maintain a suitable conformation of the parent complementarity determining region (CDR). At the same time, in order to avoid protein heterogeneity possibly caused by deamination of the NG site in the hypervariable region (CDR) of the antibody, the heavy chain variable region of the humanized antibody was designed for mutations. As a result, three humanized heavy chain variable region mutants S24B8VH-h1 (SEQ ID NO: 15), S24B8VH-h2 (SEQ ID NO: 16), S24B8VH-h3 (SEQ ID NO: 17) and one humanized light chain variable region mutant were obtained S24B8VK-h1 (SEQ ID NO: 18).

8.2. Humanization of the mouse monoclonal antibody S22E2 The humanization of the mouse monoclonal antibody S22E2 adapted the classic transplantation framework strategy. Referring to Example 8.1, grafting of the S22E2 light and heavy chain variable region CDRs resulted in the humanized heavy chain variable region mutant S22E2VH-h1 (SEQ ID NO: 19) and the humanized light chain variable region mutant S22E2VK-h1 (SEQ ID NO: 20) .

The antibody variable region genes were designed and synthesized according to the amino acid sequences of the humanized antibodies S24B8 and S22E2, and cloned into suitable eukaryotic gene expression vectors. Intact antibodies were prepared in accordance with Example 1. Three humanized variants of S24B8 were named S24B8-h1, S24B8-h2 and S24B8-h3. The humanized version of S22E2 is called S22E2-h1.

8.3. Affinity analysis of humanized S24B8 and S22E2

With reference to Example 3.2, the binding affinity between the humanized anti-TT-Hc antibody and TT-Hc was determined by Biacore XI00 using a human antibody immobilization kit (BR-1008-39). The fitting results are presented in Table 2. All heavy chain constant regions of the individual humanized antibodies were of the human IgG1 subtype.

Example 9: Preparation of bispecific antibodies

Based on the superior neutralizing activity of the combination of anti-tetanus toxin monoclonal antibodies S24B8 and S22E2 compared to treatments with each alone, antigen-binding fragments binding to different epitopes of the non-tetanus toxin protein from S24B8-h3 and S22E2-h1 were constructed as Fabs and scFvs. accordingly, to construct a human IgG1 heterodimer based on KIH (knob-in-hollow) technology. That is, the S24B8-h3-derived antigen binding fragment in the form of a Fab was fused to the N-terminus of an Fc segment containing the Knob mutation (FcK, SEQ ID NO: 40), and the S22E2-h1-derived antigen binding fragment in the form of a scFv was fused to the N-terminus of an Fc segment containing a Hole mutation (FcH, SEQ ID NO: 41), thereby constructing a bispecific antibody protein against tetanus toxin (Hc).

Three constructed eukaryotic expression vectors expressing S22E2-h1-scFv-FcHl, S24B8VH-h3+CH-IgG1K and S24B8VK-h1+CK, respectively, were cotransfected into HEK293F cells using liposomes, and the cells were cultured in suspension in serum-free medium for 3 -5 days. The culture supernatant was collected by centrifugation. Bispecific antibodies in the cultural supernatant were purified using a Protein A/G affinity chromatography column (eg, Mabselect SURE, GE Inc.). The recombinant antibody preservation buffer was then replaced with PBS buffer (pH 7.0) or other suitable buffers using a desalting column (eg, Hitrap desaulting, GE Inc.). The desalted protein solution was purified by gel filtration chromatography (SEC) using Superdex 200 (GE), thereby obtaining the bispecific antibody S22E2h1-scFv-FcH1+S24B8h3-IgG1K. If necessary, antibody samples can be sterilized by filtration and then stored in aliquots at -20°C.

Example 10: Functional Validation of Bispecific Antibodies

10.1. Inhibition of TT-Hc binding to gangliosides

With reference to Example 5, the inhibitory abilities of anti-TT-Hc antibodies on the binding of TT-Hc to the three gangliosides GD3, GT1b and GM1a were determined. The ELISA results (Figures 6, 7 and 8) demonstrated that the monoclonal antibody S22E2-h1-scFv-FcHl and the bispecific antibody S22E2h1-scFv-FcH1+S24B8h3-IgG1K had significant inhibitory effects on the binding of TT-Hc to these three gangliosides, and monoclonal antibody S24B8-h3-IgG1 did not affect the binding of TT-Hc to these three gangliosides.

10.2. Neutralizing activity

With reference to Example 6, the injected dose of tetanus toxin was 20×LD ₅₀ /mouse. The injected dose of monoclonal antibody S24B8-h3 or S22E2-h1, when used separately, was 10 μg/mouse. The injected dose of monoclonal antibody S24B8-h3 and S22E2-h1 in combination was 1.2 μg/mouse for each antibody. The injected dose of the bispecific antibody S22E2h1-scFv-FcH1+S24B8h3-IgG1K was 2.4 μg/mouse. Balb/c mice injected with 20×LD ₅₀ tetanus toxin were used as a control group. There were a total of 5 groups with 8 mice per group. Tetanus toxin was diluted to 800×LD ₅₀ /ml with boric acid buffer (ie 20×LD ₅₀ per 50 μl injection after mixing with antibody in equal volumes). S22E2 and S24B8 antibodies were diluted to 0.4 mg/ml (or 0.096 mg/ml) with boric acid buffer (i.e., for S22E2 or S24B8 treatment, 10 μg of antibody per 50 μl injection after mixing with equal volumes of tetanus toxin; and for treatment with S24B8 and S22E2 in combination, 1.2 μg of each antibody per 50 μl injection after mixing the mixture of the two antibodies (mixed in equal volumes) with tetanus toxin in equal volumes). The bispecific antibody S22E2h1-scFv-FcH1+S24B8h3-IgG1K was diluted to 0.096 mg/ml with boric acid buffer (i.e., 2.4 μg of bispecific antibody per 50 μl injection after mixing with equal volumes of tetanus toxin). Each animal was injected with 50 μl of a mixture of tetanus toxin and antibody. The experimental results are shown in Fig. 9. All Balb/c mice in the control group died from tetanus within 3 days after administration, and all Balb/c mice in the group administered the combination of S24B8-h3 and S22E2-h1 and Balb/c mice in the group administered the bispecific antibody S22E2h1-scFv -FcH1+S24B8h3-IgG1K survived at the end of the experimental period. The neutralizing activity of the bispecific antibody S22E2h1-scFv-FcH1+S24B8h3-IgG1K was superior to the neutralizing activity of monoclonal antibodies.

Example 11: Determination of Antibody Titers

The titer of the bispecific antibody S22E2h1-scFv-FcH1+S24B8h3-IgG1K was determined based on the method for determining the neutralizing activities of antibodies in Example 6. The number of international units (IU) of anti-tetanus toxin antibody contained in 1 mg of the test sample was calculated by comparing the test sample and the standard sample . Tetanus toxin dilution involved diluting the tetanus toxin with boric acid buffer to 40 test doses (L+1/10) per milliliter (i.e., 1 test dose (L+1/10) per 50 μl injection after mixing with equal amounts of antibody volumes). Dilution of the human tetanus immunoglobulin standard involved diluting the human tetanus immunoglobulin standard with boric acid buffer to 4 IU/ml (ie, 1/10 IU per 50 μl injection after mixing with equal volumes of toxin). The bispecific antibody S22E2h1-scFv-FcH1+S24B8h3-IgG1K was serially diluted with boric acid-based buffer to five concentrations at 15% intervals, i.e. 14.40 µg/ml, 16.80 µg/ml, 20 µg/ml, 23.6 µg/ml and 27.6 µg/ml (i.e. 0.36 µg, 0.42 µg, 0.50 µg, 0.59 µg and 0.69 µg of bispecific antibodies per 50 µl injection after mixing with the toxin in equal volumes). Diluted standard solutions of human tetanus immunoglobulin and solutions of bispecific antibody at various dilutions were respectively mixed with an equal volume of diluted tetanus toxin. Immediately after incubation for 1 hour at 37°C, the mixtures were injected into mice with an intramuscular injection volume of 50 μl per mouse. Mice were monitored once daily to record illness or death. The results demonstrated that all Balb/c mice in the control group died of tetanus within 72 hours after administration, and the highest dose of bispecific antibody in the group at which mice died at the same time as mice in the control group was 0.42 μg, calculated titer for which it was 238 IU/mg.

Although the present invention has been described in detail with reference to the general description and specific examples, those skilled in the art will understand that certain modifications and improvements can be made to the present invention. Accordingly, such modifications and improvements made without departing from the spirit of the invention fall within the scope of the claims.

LINKS

1. Thwaites CL, Beeching NJ, Newton CR (2015) Maternal and neonatal tetanus. Lancet 385:362–370.

2. Yousefi M, Tahmasebi F, Younesi V, Razavi A, Khoshnoodi J, Bayat AA, Abbasi E, Rabbani H, Jeddi-tehrani M, Shokri F (2014a) Characterization of neutralizing monoclonal antibodies directed against tetanus toxin fragment C. J hnmunotoxicol 11:28 34.

3. Ashton AC, Li Y, Doussau F, Weller U, Dougan G, Poulain B, Dolly JO (1995) Tetanus toxin inhibits neuroexocytosis even when its Zn21-dependent protease activity is removed. J Biol Chem 270:31386 31390.

4. Scott N, Qazi O, Wright MJ, Fairweather NF, Deonarain MP (2010) Characterization of a panel of anti-tetanus toxin single-chain Fvs reveals cooperative binding. Mol Immunol 47:1931 1941.

5. Yousefi M, Khosravi-Eghbal R, Reza Mahmoudi A, Jeddi-Tehrani M, Rabbani H, Shokri F (2014b) Comparative in vitro and in vivo assessment of toxin neutralization by antitetanus toxin monoclonal antibodies. Hum Vaccin Immunother 10:344–351.

6. Petrusic V, Zivkovic I, Stojanovic M, Stojicevic I, Marinkovic E, Dimitrijevic L (2012) Production, characterization and applications of a tetanus toxin specific monoclonal antibody T-62. Acta Histochem 114:480–486.

7. Lang AB, Cryz SJ Jr, Schurch U, Ganss MT, Bruderer U (1993immunotherapy with human monoclonal antibodies. Fragment A specificity of polyclonal and monoclonal antibodies is crucial for full protection against tetanus toxin. J Immunol 151:466 472.

--->

LIST OF SEQUENCES

<110> Beijing Wisdomab Biotechnology Co., Ltd

GENRIX (Shanghai) Biopharmaceutical Co.Ltd.

Chongqing Genrix Biopharmaceutical Co., Ltd.

<120> ANTIBODY AGAINST TETANUS TOXIN AND ITS APPLICATION

<150>CN 202011128926.0

<151> 2020-10-21

<160> 41

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 1

Ser Tyr Trp Ile Tyr

15

<210> 2

<211> 17

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 2

Glu Ile Asn Pro Thr Asn Gly Phe Ala Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Thr

<210> 3

<211> 17

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 3

Glu Ile Asn Pro Thr Ala Gly Phe Ala Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Thr

<210> 4

<211> 17

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 4

Glu Ile Asn Pro Thr Asn Ala Phe Ala Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Thr

<210> 5

<211> 6

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 5

His Phe Arg Phe Pro Tyr

15

<210> 6

<211> 11

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 6

Arg Ala Ser Gln Asp Ile Gly Ser Ser Leu Thr

1 5 10

<210> 7

<211> 7

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 7

Ala Thr Ser Ser Leu Asp Ser

15

<210> 8

<211> 9

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 8

Leu Gln Tyr Ala Ser Ser Pro Tyr Thr

15

<210> 9

<211> 5

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 9

Asp Tyr Gly Val Asn

15

<210> 10

<211> 16

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 10

Met Ile Trp Ser Asp Gly Thr Thr Asp Tyr Ser Ser Ala Leu Lys Ser

1 5 10 15

<210> 11

<211> 12

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 11

Val Asp Gly Tyr Ser His Tyr Tyr Ala Met Asp Tyr

1 5 10

<210> 12

<211> 11

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 12

Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala

1 5 10

<210> 13

<211> 7

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 13

Asn Ala Lys Thr Leu Ala Glu

15

<210> 14

<211> 9

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 14

Gln His His Tyr Gly Leu Pro Phe Thr

15

<210> 15

<211> 115

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 15

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn Pro Thr Asn Gly Phe Ala Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Thr Lys Ala Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ile His Phe Arg Phe Pro Tyr Trp Gly Gln Gly Thr Met Val Thr

100 105 110

Val Ser Ser

115

<210> 16

<211> 115

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 16

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn Pro Thr Ala Gly Phe Ala Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Thr Lys Ala Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ile His Phe Arg Phe Pro Tyr Trp Gly Gln Gly Thr Met Val Thr

100 105 110

Val Ser Ser

115

<210> 17

<211> 115

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 17

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn Pro Thr Asn Ala Phe Ala Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Thr Lys Ala Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ile His Phe Arg Phe Pro Tyr Trp Gly Gln Gly Thr Met Val Thr

100 105 110

Val Ser Ser

115

<210> 18

<211> 107

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 18

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser

20 25 30

Leu Thr Trp Leu Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Ala Ser Ser Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 19

<211> 120

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 19

Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Tyr

20 25 30

Gly Val Asn Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Met Ile Trp Ser Asp Gly Thr Thr Asp Tyr Ser Ser Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Val Asp Gly Tyr Ser His Tyr Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 20

<211> 107

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 20

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ala Pro Gln Leu Leu Ile

35 40 45

Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Ser Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Leu Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 21

<211> 445

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 21

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn Pro Thr Asn Gly Phe Ala Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Thr Lys Ala Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ile His Phe Arg Phe Pro Tyr Trp Gly Gln Gly Thr Met Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys

340 345 350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 22

<211> 445

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 22

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn Pro Thr Ala Gly Phe Ala Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Thr Lys Ala Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ile His Phe Arg Phe Pro Tyr Trp Gly Gln Gly Thr Met Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys

340 345 350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 23

<211> 445

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 23

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn Pro Thr Asn Ala Phe Ala Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Thr Lys Ala Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ile His Phe Arg Phe Pro Tyr Trp Gly Gln Gly Thr Met Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys

340 345 350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 24

<211> 214

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 24

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser

20 25 30

Leu Thr Trp Leu Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Ala Ser Ser Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 25

<211> 474

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 25

Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Tyr

20 25 30

Gly Val Asn Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Met Ile Trp Ser Asp Gly Thr Thr Asp Tyr Ser Ser Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Val Asp Gly Tyr Ser His Tyr Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser

130 135 140

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala

145 150 155 160

Ser Glu Asn Ile Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Gln Gly

165 170 175

Lys Ala Pro Gln Leu Leu Ile Tyr Asn Ala Lys Thr Leu Ala Glu Gly

180 185 190

Val Ser Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu

195 200 205

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln

210 215 220

His His Tyr Gly Leu Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu

225 230 235 240

Ile Lys Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Gln Glu Glu

370 375 380

Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

420 425 430

Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465 470

<210> 26

<211> 451

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 26

Lys Asn Leu Asp Ala Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile

1 5 10 15

Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile

20 25 30

Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala

35 40 45

Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn

50 55 60

Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn

65 70 75 80

Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys

85 90 95

Val Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile

100 105 110

Ile Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser

115 120 125

Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala

130 135 140

Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn

145 150 155 160

Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp Arg

165 170 175

Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly Ser Ala

180 185 190

Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn Ile Thr Leu

195 200 205

Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val Ser Ile Asp Lys

210 215 220

Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile Glu Lys Leu

225 230 235 240

Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe Trp Gly Asn

245 250 255

Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro Val Ala Ser Ser

260 265 270

Ser Lys Asp Val Gln Leu Lys Asn Ile Thr Asp Tyr Met Tyr Leu Thr

275 280 285

Asn Ala Pro Ser Tyr Thr Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg

290 295 300

Leu Tyr Asn Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn

305 310 315 320

Glu Ile Asp Ser Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val

325 330 335

Ser Tyr Asn Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn

340 345 350

Ala Phe Asn Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro

355 360 365

Gly Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu

370 375 380

Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser

385 390 395 400

Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro Asn

405 410 415

Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu Lys Asp

420 425 430

Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly Trp

435 440 445

Thr Asn Asp

450

<210> 27

<211> 6

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 27

His His His His His

15

<210> 28

<211> 330

<212>PRT

<213> Homo sapiens

<400> 28

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 29

<211> 330

<212>PRT

<213> Mus musculus

<400> 29

Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly

1 5 10 15

Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys

100 105 110

Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro

115 120 125

Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp

145 150 155 160

Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg

165 170 175

Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln

180 185 190

His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn

195 200 205

Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly

210 215 220

Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu

225 230 235 240

Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met

245 250 255

Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu

260 265 270

Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe

275 280 285

Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn

290 295 300

Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr

305 310 315 320

Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

325 330

<210> 30

<211> 330

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 30

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 31

<211> 330

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 31

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 32

<211> 330

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 32

Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly

1 5 10 15

Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys

100 105 110

Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro

115 120 125

Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp

145 150 155 160

Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg

165 170 175

Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln

180 185 190

His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn

195 200 205

Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly

210 215 220

Ser Val Arg Ala Pro Gln Val Cys Val Leu Pro Pro Pro Glu Glu Glu

225 230 235 240

Met Thr Lys Lys Gln Val Thr Leu Ser Cys Ala Val Thr Asp Phe Met

245 250 255

Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu

260 265 270

Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe

275 280 285

Met Val Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn

290 295 300

Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr

305 310 315 320

Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

325 330

<210> 33

<211> 330

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 33

Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly

1 5 10 15

Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys

100 105 110

Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro

115 120 125

Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp

145 150 155 160

Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg

165 170 175

Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln

180 185 190

His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn

195 200 205

Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly

210 215 220

Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Cys Glu Glu Glu

225 230 235 240

Met Thr Lys Lys Gln Val Thr Leu Trp Cys Met Val Thr Asp Phe Met

245 250 255

Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu

260 265 270

Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe

275 280 285

Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn

290 295 300

Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr

305 310 315 320

Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

325 330

<210> 34

<211> 107

<212>PRT

<213> Homo sapiens

<400> 34

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 35

<211> 106

<212>PRT

<213> Homo sapiens

<400> 35

Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser

1 5 10 15

Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp

20 25 30

Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro

35 40 45

Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn

50 55 60

Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys

65 70 75 80

Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val

85 90 95

Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210> 36

<211> 107

<212>PRT

<213> Mus musculus

<400> 36

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210> 37

<211> 106

<212>PRT

<213> Mus musculus

<400> 37

Gly Gln Pro Lys Ser Ser Pro Ser Val Thr Leu Phe Pro Pro Ser Ser

1 5 10 15

Glu Glu Leu Glu Thr Asn Lys Ala Thr Leu Val Cys Thr Ile Thr Asp

20 25 30

Phe Tyr Pro Gly Val Val Thr Val Asp Trp Lys Val Asp Gly Thr Pro

35 40 45

Val Thr Gln Gly Met Glu Thr Thr Gln Pro Ser Lys Gln Ser Asn Asn

50 55 60

Lys Tyr Met Ala Ser Ser Tyr Leu Thr Leu Thr Ala Arg Ala Trp Glu

65 70 75 80

Arg His Ser Ser Tyr Ser Cys Gln Val Thr His Glu Gly His Thr Val

85 90 95

Glu Lys Ser Leu Ser Arg Ala Asp Cys Ser

100 105

<210> 38

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic polynucleotide

<400> 38

tgcatttgaa ctccttgcc

19

<210> 39

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic polynucleotide

<400> 39

ccatcaatct tccacttgac

20

<210> 40

<211> 232

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 40

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr

130 135 140

Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 41

<211> 232

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic polypeptide

<400> 41

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Gln Glu Glu Met Thr

130 135 140

Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<---

Claims (9)

1. A monoclonal antibody that binds to tetanus toxin, containing: HCDR1 having the amino acid sequence SYWIY (SEQ ID NO: 1), HCDR2 having the amino acid sequence EINPTNGFANYNEKFKT (SEQ ID NO: 2), or EINPTAGFANYNEKFKT (SEQ ID NO: 3), or EINPTNAFANYNEKFKT (SEQ ID NO: 4), HCDR3, having the amino acid sequence HFRFPY (SEQ ID NO: 5), LCDR1 having the amino acid sequence RASQDIGSSLT (SEQ ID NO: 6), LCDR2 having the amino acid sequence ATSSLDS (SEQ ID NO: 7), and LCDR3 having the amino acid sequence LQYASSPYT (SEQ ID NO: 8); wherein the amino acid sequences HCDR and LCDR are determined according to Kabat. 2. Monoclonal antibody according to claim 1, where said monoclonal antibody contains: the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 15 and the light chain variable region amino acid sequence set forth in SEQ ID NO: 18; or the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 16 and the light chain variable region amino acid sequence set forth in SEQ ID NO: 18; or the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 17 and the light chain variable region amino acid sequence set forth in SEQ ID NO: 18. 3. Use of a monoclonal antibody according to claim 1 or 2 in the manufacture of a medicinal product for the prevention or treatment of tetanus. 4. A method for preventing or treating tetanus, comprising administering to a subject in need thereof a monoclonal antibody according to claim 1 or 2.