Antibodies that bind to tetanus toxin and uses thereof

Abstract

The present invention relates to antibodies and antigen-binding fragments thereof that bind to tetanus toxin. The present invention also relates to nucleic acids encoding such antibodies and antibody fragments, and cells expressing same. Furthermore, the present invention relates to the use of antibodies and antibody fragments in the diagnosis, prevention and treatment of Clostridium tetani or tetanus infection.

Description

Detailed Description of the Invention

[0001] The present invention relates to the field of antibodies against tetanus, in particular antibodies which bind to tetanus toxin. The present invention also relates to the use of such antibodies, for example in methods for the prevention and treatment of Clostridium tetani infection or tetanus.

[0002] Tetanus is caused by infection with Clostridium tetani spores. The spores of this bacterium are ubiquitous in the environment, for example in soil, saliva, dust, and feces. They enter the body through deep cuts, wounds, and burns that affect the nervous system. In most cases, the disease develops within 14 days of infection. Symptoms include jaw spasms and inability to open the mouth, frequent muscle spasms in the back, abdomen, and limbs, sudden painful muscle spasms often triggered by sudden noises, seizures, headaches, fever, and sweating. Tetanus can be prevented by a potent vaccine based on tetanus toxoid, but antibodies against the toxin decrease after the age of 40 / 50. Tetanus remains a significant public health problem in many parts of the world, especially in low-income countries and areas where vaccination coverage is low and unclean birth practices are common. The WHO estimates that 25,000 newborns died from neonatal tetanus in 2018.

[0003] Tetanus is caused by tetanus toxin (also called "tetanus neurotoxin" or TeNT), which is produced by Clostridium tetani under anaerobic conditions. Tetanus toxin is a highly potent neurotoxin that is expressed in the LD 50 The tetanus toxin is encoded in bacteria by the tetX gene and is expressed as a 150 kDa preprotein that is cleaved by bacterial or host proteases into two parts, a 100 kDa heavy chain and a 50 kDa light chain. Both chains are connected by a single disulfide bond. The light chain is Zn 2+ It is a metalloprotease, while the heavy chain has an N-terminal translocation domain (HN) and a C-terminal receptor binding domain (HC).

[0004] When a patient presents with a necrotic wound in a hospital emergency room, the immediate treatment, in addition to aggressive wound care, medications to control muscle spasms, and antibiotics, is usually the administration of a polyclonal human antibody, isolated from a hyperimmune human donor and known as tetanus immune globulin (TIG). In low-income countries, hyperimmune horse serum is often used, which can cause dangerous hypersensitivity reactions.

[0005] However, polyclonal TIG has various drawbacks. Due to the low percentage of antibodies specifically directed against tetanus toxin, large amounts of protein usually need to be injected. As a result, side effects such as angioedema or anaphylaxis can occur. In addition, although intrathecal administration of antibodies seems to be more effective, this method is not feasible with TIG, as it is limited by the total amount of protein that can be injected. Furthermore, neutralization efficacy varies between different lots of TIG, and there is a risk of contamination with unknown viral or blood proteins. Furthermore, TIG contains small amounts of IgA, which may trigger immune reactions in patients with IgA deficiency.

[0006] In view of the above, it is an object of the present invention to overcome the shortcomings of the prior art. In particular, it is an object of the present invention to provide an antibody that specifically binds to tetanus toxin with high binding affinity, thereby allowing for very potent antibodies to be injected in small amounts.

[0007] This object is achieved by the objects and problems described below and in the appended claims.

[0008] The present invention will be described in detail below, but it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein. It should also be understood that the terms used herein are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0009] The elements of the present invention are described below. Although these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The various described examples and embodiments should not be interpreted to limit the present invention to only the embodiments explicitly described. This specification should be understood to support and include embodiments that combine the explicitly described embodiments with any number of the disclosed elements. Furthermore, any permutation and combination of all described elements in this application should be considered to be disclosed by the description of this application, unless the context indicates otherwise.

[0010] Throughout this specification and the claims that follow, unless the context dictates otherwise, the term "comprise", as well as variations such as "comprises" and "comprising", are understood to mean the inclusion of the stated elements, integers, or steps, but not the exclusion of other unstated elements, integers, or steps. The term "consist of" is a specific embodiment of the term "comprise", which excludes other unstated elements, integers, or steps. In the context of the present invention, the term "comprise" encompasses the term "consist of". Thus, the term "comprising" encompasses "including" as well as "consisting", e.g., a composition "comprising" X may include only X or may include something additional, e.g., X+Y.

[0011] The terms "a" and "an" and "the" and similar references used in the context of describing the present invention (especially in the context of the claims) are to be construed as covering both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method for individually referring to each individual value falling within the range. Unless otherwise stated herein, each individual value is incorporated herein as if it were individually set forth herein. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0012] The term "substantially" does not exclude "completely", e.g. a composition that is "substantially free" of Y may be completely free of Y.

[0013] The term "about" in reference to a numerical value x means x±10%, for example x±5%, or x±7%, or x±10%, or x±12%, or x±15%, or x±20%.

[0014] As used herein, the term "disease" is intended to be generally synonymous with, and is used interchangeably with, the terms "disorder" and "condition" (in medical conditions), in that it refers to any abnormal condition of the human or animal body, or parts thereof, that impairs normal functioning, is typically manifested by characteristic signs and symptoms, and results in a decrease in the duration or quality of the human or animal's life.

[0015] As used herein, reference to "treatment" of a subject or patient is intended to include prevention, prophylaxis, attenuation, amelioration and cure. As used herein, the terms "subject" or "patient" are used interchangeably to mean all mammals, including humans. Examples of subjects include humans, cows, dogs, cats, horses, goats, sheep, pigs, and rabbits. In some embodiments, the patient is a human.

[0016] Dosages are often expressed relative to body weight. Thus, a dose expressed in [g, mg, or other unit] / kg (or g, mg, etc.) usually refers to [g, mg, or other unit] "per kg (or g, mg, etc.) of body weight", even if the term "body weight" is not explicitly mentioned.

[0017] The term "bind" and similar references generally mean "to specifically bind" and do not encompass non-specific attachment.

[0018] As used herein, the term "antibody" encompasses various forms of antibodies, including, but not limited to, whole antibodies, antibody fragments (such as antigen-binding fragments), human antibodies, chimeric antibodies, humanized antibodies, recombinant antibodies, genetically engineered antibodies (mutant antibodies or mutants), so long as the characteristic properties according to the invention are retained. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a monoclonal antibody. For example, the antibody is a human monoclonal antibody.

[0019] As mentioned above, the term "antibody" also generally includes antibody fragments. An antibody fragment can retain the antigen-binding activity of the antibody. Such fragments are referred to as "antigen-binding fragments". Antigen-binding fragments include, but are not limited to, single chain antibodies, Fab, Fab', F(ab')2, Fv or scFv. An antibody fragment can be obtained from an antibody by methods including digestion with enzymes such as pepsin or papain, and / or cleavage of disulfide bonds by chemical reduction. Alternatively, an antibody fragment can be obtained by recombinant means, for example, cloning and expressing a portion (fragment) of the heavy and / or light chain sequence. The present invention also encompasses single chain Fv fragments (scFv) derived from the heavy and light chains of the antibody of the present invention. For example, the present invention includes scFvs that include CDRs from the antibody of the present invention. Also included are heavy or light chain monomers and dimers, single domain heavy chain antibodies, single domain light chain antibodies, and single chain antibodies, for example single chain Fvs in which the heavy and light chain variable domains are linked by a peptide linker. The antibody fragments of the present invention may be included in various structures known to those skilled in the art. Furthermore, the sequences of the present invention may be components of multispecific molecules in which the sequences of the present invention target the epitopes of the present invention and other regions of the molecule bind to other targets. Although the present specification, including the claims, explicitly refers to antigen-binding fragments, antibody fragments, antibody variants and / or derivatives, the sequences of the present invention may be components of multispecific molecules in which the sequences of the present invention target the epitopes of the present invention and other regions bind to other targets. It is understood that the term "antibody" includes all categories of antibodies, i.e., antigen-binding fragments, antibody fragments, antibody variants and derivatives.

[0020] Human antibodies are known in the art (van Dijk, MA, and van de Winkel, JG, Curr.Opin.Chem.Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable of producing a full repertoire of human antibodies, or a selection of them, upon immunization in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al., Proc.Natl. Acad.Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Bruggemann, M., et al., Year Immunol.7 (1993) 3340). Human antibodies can also be produced in phage display libraries (Hoogenboom, HR, and Winter, G., J. Mol.Biol. 227 (1992) 381-388; Marks, JD, et al., J. Mol.Biol.222(1991)581-597). The techniques of Cole et al. and Boerner et al. can also be used to prepare human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner, P., et al., J. Immunol. 147 (1991) 86-95).In some embodiments, human monoclonal antibodies are prepared using improved EBV-B cell immortalization as described in Traggiai E, Becker S, Subbarao K, Kolesnikova L, Uematsu Y, Gismondo MR, Murphy BR, Rappuoli R, Lanzavecchia A. (2004):An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat Med. 10(8):871-5. As used herein, the term "variable region" refers to the variable region (V) of the light chain. L ), the variable region of the heavy chain (V H )) indicate each of the light and heavy chain pairs that are directly involved in the binding of the antibody to an antigen.

[0021] The antibodies of the invention can be of any isotype (e.g., IgA, IgG, IgM, i.e., α, γ or μ heavy chains). For example, the antibodies are of the IgG type. Within the IgG isotype, the antibodies may be of the IgG1, IgG2, IgG3 or IgG4 subclass, e.g., IgG1. The antibodies of the invention may have a κ or λ light chain. In some embodiments, the antibodies are of the IgG1 type and have a κ light chain.

[0022] Antibodies according to the invention may be provided in purified form. Typically, the antibodies are present in a composition that is substantially free of other polypeptides, e.g., less than 90% (by weight) of the composition is made up of other polypeptides, usually less than 60%, more usually less than 50%.

[0023] The antibodies according to the invention may be immunogenic in humans and / or non-human (or heterologous) hosts, such as mice. For example, the antibodies may have idiotopes that are immunogenic in non-human hosts but not in human hosts. Antibodies of the invention for use in humans include those that cannot be readily isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals, and generally cannot be obtained from humanized or xeno-mouse.

[0024] As used herein, a "neutralizing antibody" is an antibody that can neutralize, i.e., prevent, prevent, reduce, inhibit or interfere with, the ability of a pathogen to initiate and / or perpetuate an infection in a host. The terms "neutralizing antibody", "an antibody that neutralizes" or "antibodies that neutralize" are used interchangeably herein. These antibodies can be used, with appropriate formulation, as prophylactic or therapeutic agents, in connection with active vaccination, as diagnostic tools, or as production tools, alone or in combination, as described herein.

[0025] As used herein, the term "mutation" relates to a change in a nucleic acid sequence and / or amino acid sequence compared to a reference sequence, e.g. a corresponding genomic sequence. For example, a mutation compared to a genomic sequence may be, for example, a (naturally occurring) somatic mutation, a spontaneous mutation, an induced mutation, e.g. induced by enzymes, chemicals or radiation, or a mutation obtained by site-directed mutagenesis (a molecular biology method for producing specific and deliberate changes in a nucleic acid sequence and / or amino acid sequence). Thus, the term "mutation" or "mutating" shall be understood to include, for example, physically mutating a nucleic acid sequence or an amino acid sequence. Mutations include substitutions, deletions, insertions of one or more nucleotides or amino acids, and inversions of multiple consecutive nucleotides or amino acids. To achieve a mutation in an amino acid sequence, a mutation may be introduced into the nucleotide sequence encoding the amino acid sequence in order to express a (recombinant) mutated polypeptide. Mutations can be achieved by altering a codon in a nucleic acid molecule that encodes a certain amino acid, e.g., by site-directed mutagenesis, to produce a codon that encodes a different amino acid, or by synthesizing a sequence variant, e.g., by knowing the nucleotide sequence of a nucleic acid molecule that encodes a polypeptide and designing the synthesis of a nucleic acid molecule that contains a nucleotide sequence that encodes a variant of the polypeptide, without the need to mutate one or more nucleotides of the nucleic acid molecule.

[0026] Throughout the text of this specification, several documents are cited. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing in this specification is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0027] It is to be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein. It is also to be understood that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to limit the scope of the present invention, which is limited only by the scope of the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0028] <Antibodies and antigen-binding fragments thereof> In a first aspect, the present invention provides an (isolated) antibody or antigen-binding fragment thereof that (specifically) binds to tetanus toxin. The present invention also provides an (isolated) antibody or antigen-binding fragment thereof that (specifically) binds to tetanus toxoid.

[0029] Tetanus toxin (also called tetanus neurotoxin, TeNT) is generally described as having three domains: a light chain (L domain), and the N-terminal (HN domain) and C-terminal (HC domain) portions of the heavy chain. The C-terminal domain (HC) of the heavy chain recognizes specific cell surface receptors and is responsible for presynaptic binding. The C-terminal domain (HC) of the heavy chain has two subdomains, the N-terminal HC-N and the C-terminal HC-C. The C-terminal subdomain HC-C contains the polysialoganglioside-binding site and the nidogen-binding site, and the N-terminal subdomain HC-N is bound to the HN domain. The N-terminal domain (HN) of the heavy chain is responsible for transporting the L domain into the cytoplasm by membrane translocation. When the tetanus toxin reaches the cytoplasm, the disulfide bond between the heavy and light chains is reduced, and the L domain (light chain) is released into the cytoplasm of the neuron. The L domain is a metalloprotease that blocks neurotransmitter release from inhibitory interneurons in the spinal cord that control the balanced contraction of efferent motor neurons.

[0030] Standard methods for assessing the binding of an antibody or antigen-binding fragment thereof according to the invention are known to those skilled in the art and include, for example, ELISA (enzyme-linked immunosorbent assay), whereby the relative affinity of antibody binding can be determined as the antibody concentration required to achieve 50% of maximal binding at saturation (EC 50 ) can be determined.

[0031] An exemplary standard ELISA can be performed as follows: ELISA plates can be coated with a sufficient amount (e.g., 1 μg / ml) of the protein / complex / particle (e.g., tetanus toxoid) to be tested for antibody binding. The plates are then incubated with the antibody to be examined. After washing, antibody binding can be revealed using a labeled antibody that recognizes the test antibody, such as, for example, goat anti-human IgG conjugated with alkaline phosphatase. The plates can then be washed and the necessary substrate (e.g., p-NPP) added, and the plate read, for example, at 405 nm. The relative affinity of antibody binding can be determined by the mAb concentration required to achieve 50% maximal binding at saturation (EC 50 ) can be determined by measuring the EC 50 Values ​​may be calculated by interpolation of binding curves fitted with a four-parameter non-linear regression with varying slope.

[0032] Generally, an antibody or antigen-binding fragment thereof according to the present invention may comprise (at least) three complementarity determining regions (CDRs) on the heavy chain and (at least) three CDRs on the light chain. Generally, the complementarity determining regions (CDRs) are hypervariable regions present in the heavy chain variable domain and the light chain variable domain. Usually, the CDRs of the light chain linked to the heavy chain of an antibody together form an antigen receptor. Usually, the three CDRs (CDR1, CDR2, CDR3) are arranged non-contiguously in the variable domain. Since an antigen receptor is usually composed of two variable domains (on two different polypeptide chains, heavy and light chains: heavy chain variable region (VH) and light chain variable region (VL)), each antigen receptor usually has six CDRs (heavy chain: CDRH1, CDRH2, CDRH3, light chain: CDRL1, CDRL2, CDRL3). For example, a classical IgG antibody molecule usually has two antigen receptors, and therefore contains 12 CDRs. The CDRs on the heavy and / or light chain may be separated by framework regions (FRs), which are regions in a variable domain that are less "variable" than the CDRs. For example, the variable region (or each variable region) is composed of four framework regions separated by three CDRs.

[0033] The heavy and light chains of an exemplary antibody of the present invention were sequenced, which contains three different CDRs on the heavy chain and three different CDRs on the light chain. The positions of the CDR amino acids are defined according to the IMGT numbering system (IMGT: http: / / www.imgt.org / ; see: Lefranc, M.-P. et al. (2009) Nucleic Acids Res. 37, D1006-D1012).

[0034] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (i) heavy chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and light chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:7, respectively; or (ii) heavy chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:7, respectively, and light chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequences of SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:7, respectively. or (iii) heavy chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity to the amino acid sequences of SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, respectively, and light chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity to the amino acid sequences of SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:18, respectively; or (iv) heavy chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity to the amino acid sequences of SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, respectively, and light chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity to the amino acid sequences of SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:18, respectively.

[0035] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (i) heavy chain CDR1, CDR2, and CDR3 sequences having at least 80% sequence identity with the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and light chain CDR1, CDR2, and CDR3 sequences having at least 80% sequence identity with the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:7, respectively; or (ii) heavy chain CDR1, CDR2, and CDR3 sequences having at least 80% sequence identity with the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:7, respectively, and light chain CDR1, CDR2, and CDR3 sequences having at least 80% sequence identity with the amino acid sequences of SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:7, respectively. or (iii) heavy chain CDR1, CDR2, and CDR3 sequences having at least 80% sequence identity with the amino acid sequences of SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, respectively, and light chain CDR1, CDR2, and CDR3 sequences having at least 80% sequence identity with the amino acid sequences of SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:18, respectively; or (iv) heavy chain CDR1, CDR2, and CDR3 sequences having at least 80% sequence identity with the amino acid sequences of SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, respectively, and light chain CDR1, CDR2, and CDR3 sequences having at least 80% sequence identity with the amino acid sequences of SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:18, respectively.

[0036] As used throughout this specification, "sequence identity" is usually calculated with respect to the full length of the reference sequence (i.e., the sequence described in this application). The percentage of identity as referred to herein can be determined by methods known in the art, such as, for example, BLAST using the default parameters specified by NCBI (National Center for Biotechnology Information; http: / / www.ncbi.nlm.nih.gov / ). [Blosum 62 matrix; gap open penalty=11 and gap extension penalty=1].

[0037] As used throughout this specification, a "sequence variant" has a modified sequence in which one or more amino acids or nucleotides in a reference sequence are deleted or substituted, and / or one or more amino acids or nucleotides are inserted into the reference sequence. As a result of the modifications, a sequence variant usually has a sequence that is at least 70% identical to the reference sequence. A variant sequence that is at least 70% identical does not have more than 30 modifications, i.e., any combination of deletions, insertions or substitutions, per 100 amino acids or nucleotides of the reference sequence. A sequence variant may be at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to a reference sequence (wherein, typically, the higher the % identity of a sequence variant to a reference sequence, the more preferred the sequence variant is). In a "sequence variant", the functionality of the reference sequence (e.g., in this case, binding to tetanus toxin) may be maintained.

[0038] Generally, substitution is usually conservative amino acid substitution, in which the substituted amino acid has similar structural or chemical properties with the corresponding amino acid in reference sequence, although non-conservative amino acid substitution is also possible.For example, conservative amino acid substitution includes: one aliphatic or hydrophobic amino acid, such as alanine, valine, leucine and isoleucine, one hydroxyl group-containing amino acid, such as serine and threonine, one acidic residue, such as glutamic acid and aspartic acid, one amide group-containing residue, such as asparagine and glutamine, one aromatic residue, such as phenylalanine and tyrosine; one basic residue, such as lysine, arginine and histidine, one small amino acid, such as alanine, serine, threonine, cysteine ​​and glycine, one small amino acid.

[0039] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include the N- or C-terminal fusion of an amino acid sequence to a reporter molecule or enzyme.

[0040] The antibody or antigen-binding fragment thereof of the present invention comprises: (i) a heavy chain CDR1, CDR2 and CDR3 sequence having at least 90% sequence identity (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and a heavy chain CDR1, CDR2 and CDR3 sequence having at least 90% sequence identity to the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:7, respectively. (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more); or (ii) heavy chain CDR1, CDR2, and CDR3 sequences having at least 90% sequence identity (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively. or (iii) light chain CDR1, CDR2, and CDR3 sequences having at least 90% sequence identity (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) with the amino acid sequences of SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:7, respectively; or (iv) light chain CDR1, CDR2, and CDR3 sequences having at least 90% sequence identity (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) with the amino acid sequences of SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, respectively. heavy chain CDR1, CDR2, and CDR3 sequences having at least 90% sequence identity (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequences of SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:18, respectively;or (iv) heavy chain CDR1, CDR2, and CDR3 sequences having at least 90% sequence identity (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequences of SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, respectively, and light chain CDR1, CDR2, and CDR3 sequences having at least 90% sequence identity (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequences of SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:18, respectively;

[0041] Thus, the antibody or antigen-binding fragment thereof preferably comprises: - the heavy chain CDR1 sequence set forth in SEQ ID NO:1; - the heavy chain CDR2 sequence set forth in SEQ ID NO:2; - the heavy chain CDR3 sequence set forth in SEQ ID NO:3; - the light chain CDR1 sequence set forth in SEQ ID NO:4; - a light chain CDR2 sequence as set forth in SEQ ID NO: 5 or 6; and - the light chain CDR3 sequence as set forth in SEQ ID NO:7.

[0042] Alternatively, the antibody or antigen-binding fragment thereof preferably comprises: - the heavy chain CDR1 sequence set forth in SEQ ID NO: 12; - the heavy chain CDR2 sequence set forth in SEQ ID NO: 13; - the heavy chain CDR3 sequence set forth in SEQ ID NO: 14; - the light chain CDR1 sequence set forth in SEQ ID NO: 15; - a light chain CDR2 sequence as set forth in SEQ ID NO: 16 or 17; and - the light chain CDR3 sequence set forth in SEQ ID NO: 18.

[0043] In some embodiments, an antibody or antigen-binding fragment thereof of the invention has: (i) 70% or more (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO:8. and a light chain variable region (VL) comprising an amino acid sequence having 70% or more (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity with SEQ ID NO: 9. Thereby, the CDR sequences defined above (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 or 6, and SEQ ID NO: 7, respectively) can be maintained.

[0044] In some embodiments, an antibody or antigen-binding fragment thereof of the invention has: (i) a sequence identical to SEQ ID NO: 19 that is 70% or more (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO: 19; and a light chain variable region comprising an amino acid sequence having 70% or more (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity with SEQ ID NO: 20, thereby maintaining the CDR sequences defined above (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 or 17, and SEQ ID NO: 18, respectively).

[0045] In some embodiments, an antibody or antigen-binding fragment thereof of the invention has (i) 75% or more (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO:8. and a light chain variable region comprising an amino acid sequence having 75% or more (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO:9, thereby maintaining the CDR sequences defined above (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO:4, SEQ ID NO:5 or 6, and SEQ ID NO:7, respectively).

[0046] In some embodiments, an antibody or antigen-binding fragment thereof of the invention has (i) 75% or more (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 19. and a light chain variable region comprising an amino acid sequence having 75% or more (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 20, thereby maintaining the CDR sequences defined above (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 or 17, and SEQ ID NO: 18, respectively).

[0047] In some embodiments, an antibody or antigen-binding fragment thereof of the invention comprises: (i) a heavy chain variable region comprising an amino acid sequence having 80% or more (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO:8; and a light chain variable region comprising an amino acid sequence having 80% or more (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO:9. This allows the CDR sequences defined above to be maintained (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO:4, SEQ ID NO:5 or 6, and SEQ ID NO:7, respectively).

[0048] In some embodiments, an antibody or antigen-binding fragment thereof of the invention comprises: (i) a heavy chain variable region comprising an amino acid sequence having 80% or more (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO:19; and a light chain variable region comprising an amino acid sequence having 80% or more (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO:20. This allows the CDR sequences defined above to be maintained (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 or 17, and SEQ ID NO: 18, respectively).

[0049] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises (i) a heavy chain variable region comprising an amino acid sequence having 85% or more (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 8, and a light chain variable region comprising an amino acid sequence having 85% or more (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 9, thereby maintaining the CDR sequences defined above (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 or 6, and SEQ ID NO: 7, respectively).

[0050] In some embodiments, an antibody or antigen-binding fragment thereof of the invention comprises: (i) a heavy chain variable region comprising an amino acid sequence having 85% or more (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO:19; and a light chain variable region comprising an amino acid sequence having 85% or more (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO:20. This allows the CDR sequences defined above to be maintained (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 or 17, and SEQ ID NO: 18, respectively).

[0051] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises (i) a heavy chain variable region comprising an amino acid sequence having 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 8, and a light chain variable region comprising an amino acid sequence having 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 9, thereby maintaining the CDR sequences defined above (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 or 6, and SEQ ID NO: 7, respectively).

[0052] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises (i) a heavy chain variable region comprising an amino acid sequence having 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 19, and a light chain variable region comprising an amino acid sequence having 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 20, thereby maintaining the CDR sequences defined above (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 or 17, and SEQ ID NO: 18, respectively).

[0053] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises (i) a heavy chain variable region comprising an amino acid sequence having 95% or more (e.g., 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 8, and a light chain variable region comprising an amino acid sequence having 95% or more (e.g., 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 9, thereby maintaining the CDR sequences defined above (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 or 6, and SEQ ID NO: 7, respectively).

[0054] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises (i) a heavy chain variable region comprising an amino acid sequence having 95% or more (e.g., 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 19, and a light chain variable region comprising an amino acid sequence having 95% or more (e.g., 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 20, thereby maintaining the CDR sequences defined above (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 or 17, and SEQ ID NO: 18, respectively).

[0055] More specifically, the antibody or antigen-binding fragment thereof preferably comprises a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:8, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:9.

[0056] Alternatively, the antibody or antigen-binding fragment thereof preferably comprises a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 19, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 20.

[0057] In some embodiments, an antibody or antigen-binding fragment thereof of the invention has: (i) 70% or more (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO:10; and a light chain comprising an amino acid sequence having 70% or more (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 11, thereby maintaining the CDR sequences defined above (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 or 6, and SEQ ID NO: 7, respectively).

[0058] In some embodiments, an antibody or antigen-binding fragment thereof of the invention has: (i) 70% or more (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO:21; and a light chain comprising an amino acid sequence having 70% or more (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 22, thereby maintaining the CDR sequences defined above (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 or 17, and SEQ ID NO: 18, respectively).

[0059] In some embodiments, an antibody or antigen-binding fragment thereof of the invention has: (i) 75% or more (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO:10; and a light chain comprising an amino acid sequence having 75% or more (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 11, thereby maintaining the CDR sequences defined above (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 or 6, and SEQ ID NO: 7, respectively).

[0060] In some embodiments, an antibody or antigen-binding fragment thereof of the invention has: (i) 75% or more (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO:21. and a light chain comprising an amino acid sequence having 75% or more (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 22, thereby maintaining the CDR sequences defined above (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 or 17, and SEQ ID NO: 18, respectively).

[0061] In some embodiments, an antibody or antigen-binding fragment thereof of the invention comprises (i) a heavy chain comprising an amino acid sequence having 80% or more (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO:10, and a light chain comprising an amino acid sequence having 80% or more (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO:11. This allows the CDR sequences defined above to be maintained (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO:4, SEQ ID NO:5 or 6, and SEQ ID NO:7, respectively).

[0062] In some embodiments, an antibody or antigen-binding fragment thereof of the invention comprises (i) a heavy chain comprising an amino acid sequence having 80% or more (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO:21, and a light chain comprising an amino acid sequence having 80% or more (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO:22. This allows the CDR sequences defined above to be maintained (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 or 17, and SEQ ID NO: 18, respectively).

[0063] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises (i) a heavy chain comprising an amino acid sequence having 85% or more (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 10, and a light chain comprising an amino acid sequence having 85% or more (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 11, thereby maintaining the CDR sequences defined above (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 or 6, and SEQ ID NO: 7, respectively).

[0064] In some embodiments, an antibody or antigen-binding fragment thereof of the invention comprises (i) a heavy chain comprising an amino acid sequence having 85% or more (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO:21, and a light chain comprising an amino acid sequence having 85% or more (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO:22. This allows the CDR sequences defined above to be maintained (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 or 17, and SEQ ID NO: 18, respectively).

[0065] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises (i) a heavy chain comprising an amino acid sequence having 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 10, and a light chain comprising an amino acid sequence having 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 11, thereby maintaining the CDR sequences defined above (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 or 6, and SEQ ID NO: 7, respectively).

[0066] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises (i) a heavy chain comprising an amino acid sequence having 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 21, and a light chain comprising an amino acid sequence having 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 22, thereby maintaining the CDR sequences defined above (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 or 17, and SEQ ID NO: 18, respectively).

[0067] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises (i) a heavy chain comprising an amino acid sequence having 95% or more (e.g., 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 10, and a light chain comprising an amino acid sequence having 95% or more (e.g., 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 11, thereby maintaining the CDR sequences defined above (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 or 6, and SEQ ID NO: 7, respectively).

[0068] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises (i) a heavy chain comprising an amino acid sequence having 95% or more (e.g., 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 21, and a light chain comprising an amino acid sequence having 95% or more (e.g., 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 22, thereby maintaining the CDR sequences defined above (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 or 17, and SEQ ID NO: 18, respectively).

[0069] More specifically, the antibody or antigen-binding fragment thereof preferably comprises a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:10, and a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:11.

[0070] Alternatively, the antibody or antigen-binding fragment thereof preferably comprises a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:21, and a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:22.

[0071] The CDR and VH / VL sequences and Fab sequences of exemplary antibodies of the invention, namely antibodies TT104 and TT110, are shown in Table 1 below. [Table 1]

[0072] In some embodiments, the antibodies of the invention are human antibodies. In some embodiments, the antibodies of the invention are monoclonal antibodies. For example, the antibodies of the invention may be human monoclonal antibodies.

[0073] In some embodiments, an antibody, or antigen-binding fragment thereof, according to the invention comprises an Fc portion. The Fc portion may be of human origin, such as from human IgG1, IgG2, IgG3, and / or IgG4, such as from human IgG1.

[0074] As used herein, the term "Fc portion" refers to a sequence derived from a portion of an immunoglobulin heavy chain beginning at the hinge region immediately upstream of the papain cleavage site (e.g., residue 216 in native IgG, with the first residue of the heavy chain constant region being 114) and ending at the C-terminus of the immunoglobulin heavy chain. Thus, the Fc portion may be a complete Fc portion or a portion thereof (e.g., a domain). A complete Fc portion includes at least the hinge domain, the CH2 domain, and the CH3 domain (e.g., EU amino acid positions 216-446). An additional lysine residue (K) may be present at the extreme C-terminus of the Fc portion, but is often cleaved from the mature antibody.

[0075] Each amino acid position within the Fc portion is numbered herein according to the art-recognized EU numbering system of Kabat, see, e.g., Kabat et al., "Sequences of Proteins of Immunological Interest", US Dept. Health and Human Services, 1983 and 1987. The EU index or EU numbering in Kabat refers to the numbering of EU antibodies (Edelman GM, Cunningham BA, Gall WE, Gottlieb PD, Rutishauser U, Waxdal MJ. The covalent structure of an entire gammaG immunoglobulin molecule. Proc Natl Acad Sci US A. 1969;63(1):78-85; Kabat EA, National Institutes of Health (US) Office of the Director, "Sequences of Proteins of Immunological Interest", 5 th edition, Bethesda, MD: US Dept. of Health and Human Services, Public Health Service, National Institutes of Health, 1991, incorporated herein by reference in its entirety).

[0076] In some embodiments, in the context of the present invention, the Fc portion comprises at least one of the hinge (e.g., upper, middle, and / or lower hinge regions), CH2, or CH3 domains, or variants, parts, or fragments thereof. The Fc portion may comprise at least the hinge, CH2, or CH3 domains. The Fc portion may be a complete Fc portion. The Fc portion may also comprise one or more amino acid insertions, deletions, or substitutions relative to a naturally occurring Fc portion. For example, at least one of the hinge, CH2, or CH3 domains (or parts thereof) may be deleted. For example, the Fc portion may comprise or consist of: (i) a hinge domain (or a portion thereof) fused to a CH2 domain (or a portion thereof), (ii) a hinge domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), (iii) a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), (iv) a hinge domain (or a portion thereof), (v) a CH2 domain (or a portion thereof), or (vi) a CH3 domain or a portion thereof.

[0077] It will be understood by those skilled in the art that the Fc portion may be modified to differ in amino acid sequence from the complete Fc portion of a naturally occurring immunoglobulin molecule, while retaining at least one desirable function conferred by the naturally occurring Fc portion. Such functions include Fc receptor (FcR) binding, antibody half-life regulation, ADCC function, Protein A binding, Protein G binding, complement fixation, etc. Some of the naturally occurring Fc portions that are responsible for and / or essential for such functions are known to those skilled in the art. In some embodiments, an antibody according to the invention comprises an (complete) Fc portion / Fc region with intact interaction / binding to FcR.

[0078] Additionally, the Fc fragments were measured by ELISA (Hessell AJ, Hangartner L, Hunter M, Havenith CEG, Beurskens FJ, Bakker JM, Lanigan CMS, Landucci G, Forthal DN, 1999). Parren PWHI, et al.: Fc receptor but not complement binding is important in antibody protection against HIV the Neonatal Fc Receptor Affects Fc Effector Functions 2015, 194:5497-5508) Perez LG, Costa MR, Todd CA, Haynes BF, Montefiori DC: Utilization of immunoglobulin G Fc receptors by J Virol 2009, 83:7397-7410: Piccoli L, Campo I, Fregni CS, Rodriguez BMF, Minola A, Sallusto F, Luisetti M, Corti D, Lanzavecchia A: Neutralization and clearance of GM-CSF by autoantibodies in pulmonary alveolar proteinosis.Nat Commun 2015, 6:1-9).

[0079] For example, FcR binding is mediated by the interaction of the Fc portion (of an antibody) with the Fc receptor (FcR), a specialized cell surface receptor on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes and the lysis of red blood cells and various other cellular targets (e.g., tumor cells) coated with the corresponding antibodies through antibody-dependent cell-mediated cytotoxicity (ADCC; Van de Winkel, JG, and Anderson, CL, J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin classes, with Fc receptors for IgG antibodies termed FcγR, those for IgE FcεR, those for IgA FcαR, and neonatal Fc receptors termed FcRn. Fc receptor binding has been disclosed, for example, in Ravetch, JV, and Kinet, JP, Annu. Rev. Immunol. 9 (1991) 457-492; Capel, PJ, et al., Immunomethods 4 (1994) 25-34; de Haas, M., et al., J Lab. Clin. Med. 126 (1995) 330-341; and Gessner, JE, et al., Ann. Hematol. 76 (1998) 231-248.

[0080] Cross-linking of receptors by the Fc domains of native IgG antibodies (FcγRs) induces a wide variety of effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity, and release of inflammatory mediators, as well as regulation of immune complex clearance and antibody production. Therefore, the Fc portion can provide cross-linking of receptors (FcγRs). In humans, three classes of FcγR have been characterized: (i) FcγRI (CD64), which binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils; (ii) FcγRII (CD32), which binds complexed IgG with medium to low affinity, is widely expressed especially on leukocytes and is known to play a central role in antibody-mediated immunity and is divided into FcγRIIA, FcγRIIB and FcγRIIC, which serve different functions in the immune system but bind IgG-Fc with similar low affinity and the ectodomains of these receptors are highly homologous; (iii) FcγRIII (CD16), which binds IgG with medium to low affinity and exists in two types: FcγRIIIA, which is present on NK cells, macrophages, eosinophils, some monocytes and T cells and mediates ADCC, and FcγRIIIB, which is highly expressed on neutrophils. FcγRIIA is present on many cells involved in killing (e.g., macrophages, monocytes, and neutrophils) and appears to be able to activate the killing process. FcγRIIB appears to be involved in inhibitory processes and is present on B cells, macrophages, mast cells, and eosinophils. Importantly, 75% of all FcγRIIB is present in the liver (Ganesan, LP et al., 2012: FcγRIIb on liver sinusoidal endothelium clears small immune complexes. Journal of Immunology 189: 4981-4988).FcγRIIB is abundantly expressed on liver sinusoidal endothelium, called LSECs, and on hepatic Kupffer cells, which are the major site of clearance of small immune complexes (Ganesan, LP et al., 2012: FcγRIIb on liver sinusoidal endothelium clears small immune complexes. Journal of Immunology 189: 4981-4988).

[0081] Thus, the antibodies and antigen-binding fragments thereof of the present invention may be capable of binding to FcγRIIb, and may include antibodies that contain an Fc portion, particularly an Fc region, for binding to FcγRIIb, such as IgG-type antibodies. Furthermore, the Fc portion may be designed to enhance FcγRIIb binding by introducing the mutations S267E and L328F, as described in Chu, SY et al., 2008: Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcγRIIb with Fc-engineered antibodies. Molecular Immunology 45, 3926-3933. This can facilitate the clearance of immune complexes (Chu, S., et al., 2014: Accelerated Clearance of IgE In Chimpanzees Is Mediated By Xmab7195, An Fc-Engineered Antibody With Enhanced Affinity For Inhibitory Receptor FcγRIIb. Am J Respir Crit, American Thoracic Society International Conference Abstracts). Thus, the antibody or antigen-binding fragment thereof of the present invention may comprise an Fc portion engineered with the mutations S267E and L328F, in particular as described in Chu, SY et al., 2008: Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcγRIIb with Fc-engineered antibodies. Molecular Immunology 45, 3926-3933.

[0082] In B cells, it appears to function to suppress further immunoglobulin production and isotype switching, for example to IgE class. In macrophages, FcγRIIB inhibits phagocytosis via FcγRIIA. In eosinophils and mast cells, type B may help suppress the activation of these cells by binding IgE to a different receptor.

[0083] With regard to FcγRI binding, modifications of at least one of E233-G236, P238, D265, N297, A327, and P329 in native IgG reduce binding to FcγRI. Substitution of IgG2 residues at positions 233-236 in IgG1 and IgG4 reduces binding to FcγRI by 10. 32-fold reduction in FcγRII binding and abolished human monocyte responses to antibody-sensitized red blood cells (Armour, KL, et al. Eur. J. Immunol. 29 (1999) 2613-2624). With regard to FcγRII binding, for example, at least one of the following IgG mutations is shown to reduce binding to FcγRIIA: E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414. With regard to FcγRIII binding, for example, at least one of the following mutations reduces binding to FcγRIIIA: E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338, D376. Mapping of the binding site on human IgG1 for Fc receptors, the above mutation sites, and methods for measuring binding to FcγRI and FcγRIIA are described in Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604. For example, single mutations (S239D or I332E), double mutations (S239D / I332E), and triple mutations (S239D / I332E / A330L) improved affinity to human FcγRIIIa. Furthermore, the addition of the mutation G236A to S239D / I332E not only improved the FcγRIIa:FcγRIIb ratio, but also enhanced binding to FcγRIIIa. Thus, it has been described that the mutation G236A / S239D / A330L / I332E enhances the binding of FcγRIIa to FcγRIIIa.

[0084] With regard to binding to FcγRII, two regions of native IgG Fc appear to be important for the interaction of FcγRII with IgG: (i) the lower hinge region of IgG Fc, specifically amino acid residues L, L, G, G (234-237, EU numbering), and (ii) the adjacent regions of the CH2 domain of IgG Fc, specifically the loops and strands of the upper CH2 domain adjacent to the lower hinge region, e.g., the region of P331 (Wines, BD, et al., J. Immunol. 2000; 164: 5313 - 5318). Furthermore, FcγRI appears to bind to the same site on IgG Fc, whereas FcRn and Protein A bind to different sites on IgG Fc, which appear to be at the CH2-CH3 interface (Wines, BD, et al., J. Immunol. 2000; 164: 5313 - 5318).

[0085] For example, the Fc portion may comprise or consist of at least a portion of an Fc portion known in the art to be required for FcRn binding or extended half-life. Alternatively or additionally, the Fc portion of an antibody of the invention comprises at least a portion of an Fc molecule known in the art to be required for Protein A binding, and / or the Fc portion of an antibody of the invention comprises at least a portion of an Fc molecule known in the art to be required for Protein G binding. The Fc portion may comprise at least a portion of an Fc molecule known in the art to be required for FcγR binding. As outlined above, the Fc portion therefore comprises at least (i) the lower hinge region of native IgG Fc, in particular amino acid residues L, L, G, G (234-237, EU numbering), and (ii) the adjacent region of the CH2 domain of native IgG Fc, in particular which may consist of the loops and strands of the upper CH2 domain adjacent to the lower hinge region, e.g. the region of P331, e.g. a region of at least 3, 4, 5, 6, 7, 8, 9, or 10 contiguous amino acids of the upper CH2 domain of native IgG Fc surrounding P331, e.g. the region between amino acids 320 and 340 (EU numbering) of native IgG Fc.

[0086] Furthermore, the antibodies according to the invention can be modified by introducing (random) amino acid mutations in specific regions of the CH2 or CH3 domain of the heavy chain to alter the binding affinity to FcR and / or serum half-life compared to the unmodified antibody. Examples of such modifications include, but are not limited to, the substitution of at least one amino acid in the heavy chain constant region selected from the group including amino acid residues 250, 314 and 428. Further examples of such Fc modifications are described in Saxena A, Wu D. Advances in Therapeutic Fc Engineering - Modulation of IgG-Associated Effector Functions and Serum Half-life. Front Immunol. 2016;7:580, which is incorporated herein by reference. In some embodiments, the antibody can include "YTE" mutations (M252Y / S254T / T256E; EU numbering). In some embodiments, the antibody can include the mutations M428L and / or N434S in the heavy chain constant region (EU numbering).

[0087] In some embodiments, an antibody, or antigen-binding fragment thereof, according to the invention comprises an Fc region. As used herein, the term "Fc region" refers to a portion of an immunoglobulin formed by two or more Fc portions of antibody heavy chains. For example, the Fc region may be a monomeric or "single-chain" Fc region (i.e., scFc region). A single-chain Fc region is composed of Fc portions linked in a single polypeptide chain (e.g., encoded in a single contiguous nucleic acid sequence). An exemplary scFc region is disclosed in WO 2008 / 143954 A2. The Fc region may be a dimer. A "dimeric Fc region" or "dcFc" refers to a dimer formed by the Fc portions of two separate immunoglobulin heavy chains. A dimeric Fc region may be a homodimer of two identical Fc portions (e.g., Fc regions of naturally occurring immunoglobulins) or a heterodimer of two non-identical Fc portions. The Fc portions of the Fc region may be of the same or different classes and / or subclasses. For example, the Fc portions may be derived from immunoglobulins of the IgG1, IgG2, IgG3 or IgG4 subclass (e.g., human immunoglobulins). The Fc portions of the Fc region may be of the same class and subclass. However, the Fc region (or one or more Fc portions of the Fc region) may also be chimeric, such that the chimeric Fc region may contain Fc portions derived from different immunoglobulin classes and / or subclasses. For example, at least two Fc portions of a dimeric or single-chain Fc region may be derived from different immunoglobulin classes and / or subclasses. Additionally or alternatively, the chimeric Fc region may contain one or more chimeric Fc portions. For example, the chimeric Fc region or portion may contain one or more portions derived from an immunoglobulin of a first subclass (e.g., IgG1, IgG2, or IgG3 subclass), while the remaining portions of the Fc region or portion are of a different subclass. For example, an Fc region or portion of an Fc polypeptide may comprise a CH2 and / or CH3 domain from an immunoglobulin of a first subclass (e.g., IgG1, IgG2 or IgG4 subclass) and a hinge region from an immunoglobulin of a second subclass (e.g., IgG3 subclass). For example, an Fc region or portion may comprise a hinge and / or CH2 domain from an immunoglobulin of a first subclass (e.g., IgG4 subclass) and a CH3 domain from an immunoglobulin of a second subclass (e.g., IgG1, IgG2 or IgG3 subclass). For example, a chimeric Fc region may comprise an Fc portion (e.g., a complete Fc portion) from an immunoglobulin of a first subclass (e.g., IgG4 subclass) and an Fc portion from an immunoglobulin of a second subclass (e.g., IgG1, IgG2 or IgG3 subclass). For example, an Fc region or portion may comprise a CH2 domain from an IgG4 immunoglobulin and a CH3 domain from an IgG1 immunoglobulin.For example, an Fc region or portion may comprise a CH1 domain and a CH2 domain derived from an IgG4 molecule, and a CH3 domain derived from an IgG1 molecule. For example, an Fc region or portion may comprise a portion of a CH2 domain from a particular subclass of antibody, such as positions 292-340 of the CH2 domain. For example, an Fc region or portion may comprise positions 292-340 of CH2 derived from the IgG4 portion and the remaining amino acids of CH2 derived from the IgG1 portion (alternatively, positions 292-340 of CH2 may be derived from the IgG1 portion and the remaining portion of CH2 may be derived from the IgG4 portion). Further, the Fc region or portion may (additionally or alternatively) comprise, for example, a chimeric hinge region. For example, the chimeric hinge may be, for example, derived in part from an IgG1, IgG2, or IgG4 molecule (e.g., upper and lower middle hinge sequences) and in part from an IgG3 molecule (e.g., middle hinge sequence). In another example, the Fc region or portion may comprise a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule. In another example, the chimeric hinge may comprise upper and lower hinge domains derived from an IgG4 molecule and a middle hinge domain derived from an IgG1 molecule. Such a chimeric hinge may be generated, for example, by introducing a proline substitution (Ser228Pro) at EU position 228 of the middle hinge domain of the IgG4 hinge region. In other embodiments, the chimeric hinge may comprise amino acids at positions EU233-236 derived from an IgG2 antibody and / or the Ser228Pro mutation, with the remaining amino acids of the hinge being derived from an IgG4 antibody. Further chimeric hinges which may be used in the Fc portion of an antibody according to the invention are described in US 2005 / 0163783 A1.

[0088] In some embodiments, the Fc portion or Fc region comprises or consists of an amino acid sequence derived from a human immunoglobulin sequence (e.g., from an Fc region or Fc portion derived from a human IgG molecule). However, the Fc portion or Fc region may also comprise one or more amino acids from other mammalian species. For example, a primate Fc portion or primate binding site may be included in the antibody or antigen-binding fragment. Alternatively, one or more mouse amino acids may be present in the Fc portion or Fc region.

[0089] In some embodiments, the antibody, or antigen-binding fragment, comprises, in addition to the Fc portion, in particular as described above, other portions derived from the constant region, in particular from the constant region of IgG, such as the constant region of (human) IgG1. The antibody according to the invention may comprise, in addition to the Fc portion, in particular as described above, all other portions of the constant region, in particular all other portions of the constant region of IgG (such as (human) IgG1). In other words, the antibody, or antigen-binding fragment, may comprise the (complete) Fc region from human IgG1. In some embodiments, the antibody, or antigen-binding fragment, comprises, in addition to the (complete) Fc region, in particular from human IgG1, also all other portions of the constant region of IgG, such as all other portions of the constant region of (human) IgG1.

[0090] In general, however, the antibodies of the invention can be of any isotype (e.g., IgA, IgG, IgM, i.e., α, γ or μ heavy chains). For example, the antibodies may be of the IgG type. Within the IgG isotype, the antibodies may be of the IgG1, IgG2, IgG3 or IgG4 subclass, e.g., IgG1. The antibodies of the invention may have a κ or λ light chain. In some embodiments, the antibodies are of the IgG1 type and have a lambda or kappa light chain.

[0091] In some embodiments, the antibody is of the human IgG1 type. The antibody may be of any allotype. The term "allotype" refers to the allelic variations found among IgG subclasses. For example, the antibody may be of the G1m1 (or G1m(a)) allotype, the G1m2 (or G1m(x)) allotype, the G1m3 (or G1m(f)) allotype, and / or the G1m17 (or Gm(z)) allotype. The G1m3 and G1m17 allotypes are located in the same position in the CH1 domain (position 214 in the EU numbering). G1m3 corresponds to R214 (EU), while G1m17 corresponds to K214 (EU). The G1m1 allotype is located in the CH3 domain (positions 356 and 358 (EU)) and refers to the substitutions E356D and M358L. G1m2 allotype refers to a substitution of alanine at position 431 (EU) with glycine. G1m1 allotype may be combined with, for example, G1m3 or G1m17 allotypes. In some embodiments, the antibody is a G1m3 allotype (G1m3,-1) that does not contain G1m1. In some embodiments, the antibody is a G1m17,1 allotype. In some embodiments, the antibody is a G1m3,1 allotype. In some embodiments, the antibody is a G1m17 allotype (G1m17,-1) that does not have G1m1. Optionally, these allotypes may be combined (or not) with G1m2, G1m27 or G1m28 allotypes. For example, the antibody may be a G1m17,1,2 allotype.

[0092] In general, the antibodies or antigen-binding fragments according to the invention may be glycosylated. For example, N-linked glycans attached to the CH2 domain of the heavy chain may affect C1q and FcR binding, resulting in glycosylated antibodies having lower affinity for these receptors. Thus, the CH2 domain of the Fc portion of the antibodies according to the invention may contain one or more mutations in which glycosylated residues are replaced with non-glycosylated residues. For example, the glycosylation of the antibody does not elicit a human immunogenic response after administration.

[0093] Exemplary sequences of the constant region are the amino acid sequences set forth in SEQ ID NOs: 23 to 26. For example, the amino acid sequence of IgG1 CH1-CH2-CH3 is that set forth in SEQ ID NO: 23 or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity (e.g., containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations). The light chain constant region may be that set forth in SEQ ID NO: 24 or 25 or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity (e.g., containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations).

[0094] As mentioned above, the present invention encompasses antigen-binding fragments. The antigen-binding fragment may or may not include a portion of the Fc portion, particularly the complete Fc portion. In some embodiments, the antibody or antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, Fv or scFv. For example, F(ab')2 (which may be obtained by pepsin cleavage or recombinant expression) and Fab' (which may be obtained from F(ab')2 or recombinant expression) usually include the hinge region.

[0095] In some embodiments, the antibody or antigen-binding fragment may be a single-chain antibody (or fragment). The single-chain antibody (or fragment) may encode a complete set of six CDRs, i.e., three heavy chain CDRs and three light chain CDRs. More specifically, the single-chain antibody (or fragment) may comprise a heavy chain variable region (VH) and a light chain variable region (VL), including, for example, the VH and VL sequences as described above.

[0096] In some embodiments, the antibody or antigen-binding fragment thereof does not include an Fc portion. In other words, the antibody or antigen-binding fragment thereof may not include any of the hinge region, the CH2 region, and the CH3 region. In one embodiment, the antibody or antigen-binding fragment thereof is a Fab. A Fab typically includes a single constant region of a heavy chain (e.g., CH1) and a light chain (e.g., CL) in addition to heavy and light chain variable domains. A Fab may be obtained, for example, by papain cleavage or by recombinant expression, for example, with a stop codon introduced after the nucleic acid sequence encoding the heavy chain VH and CH1 region (particularly immediately after the CH1 coding sequence). For example, the amino acid sequence of the heavy chain constant region (CH1 region) of the Fab may be as set forth in SEQ ID NO: 26, or a sequence variant thereof (e.g., including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity. The light chain constant region of a Fab will usually correspond to that of an "intact" immunoglobulin and therefore may be as set forth in SEQ ID NO: 24 or 25; or a sequence variant thereof (e.g., containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity.

[0097] In some embodiments, the Fab comprises or consists of a heavy chain sequence set forth in SEQ ID NO: 10, or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity (e.g., containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations); and a light chain sequence set forth in SEQ ID NO: 11, or a sequence variant thereof having at least 70%, at least 75%, at least 90%, at least 95% or at least 99% sequence identity (e.g., containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations). This allows the CDR sequences defined above to be maintained (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO:4, SEQ ID NO:5 or 6, and SEQ ID NO:7, respectively).

[0098] In other embodiments, the Fab comprises or consists of a heavy chain sequence set forth in SEQ ID NO:21, or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity (e.g. containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations); and a light chain sequence set forth in SEQ ID NO:22, or a sequence variant thereof having at least 70%, at least 75%, at least 90%, at least 95% or at least 99% sequence identity (e.g. containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations). Thereby, the CDR sequences defined above (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 or 17, and SEQ ID NO: 18, respectively) can be maintained.

[0099] The antibodies of the present invention also include hybrid antibody molecules that contain the six CDRs from an antibody of the present invention as defined above and one or more CDRs from another antibody against the antigen. For example, the antibody may be bispecific.

[0100] Mutant antibodies are also within the scope of the present invention. Thus, variants of the sequences described herein are also within the scope of the present invention. Such variants include naturally occurring variants that arise by somatic mutation in vivo during an immune response or in vitro with the culture of immortalized B cell clones. Alternatively, variants may arise due to the degeneracy of the genetic code, or may arise due to transcription or translation errors.

[0101] The antibodies or antigen-binding fragments thereof of the present invention may be provided in purified form. Typically, the antibodies or antigen-binding fragments are present in a composition that is substantially free of other polypeptides, e.g., less than 90% (by weight) of the composition is made up of other polypeptides, usually less than 60%, more usually less than 50%.

[0102] The antibodies of the present invention may be immunogenic in a non-human host (or a heterologous host), such as a mouse. In particular, the antibodies may have idiotopes that are immunogenic in a non-human host but not in a human host. In particular, the antibodies of the present invention for use in humans include those that cannot be easily isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals, and generally cannot be obtained from humanized or xeno-mouse.

[0103] <Nucleic acid> In another aspect, the present invention also provides a nucleic acid molecule comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof according to the present invention as described above.

[0104] In some embodiments, the nucleic acid molecule comprises one or more polynucleotides encoding an exemplary antibody of the invention (e.g., as described in Table 1 above), or a sequence variant thereof as described herein (e.g., having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity as described above).

[0105] Examples of nucleic acid molecules and / or polynucleotides include, for example, recombinant polynucleotides, vectors, oligonucleotides, RNA molecules such as rRNA, mRNA, miRNA, siRNA, or tRNA, or DNA molecules such as cDNA. The nucleic acid can encode the light chain and / or the heavy chain of the antibody. In other words, the light chain and the heavy chain of the antibody may be encoded by the same nucleic acid molecule (e.g., in a bicistronic manner). Alternatively, the light chain and the heavy chain of the antibody may be encoded by separate nucleic acid molecules.

[0106] Due to the redundancy of the genetic code, the present invention also includes sequence variants of nucleic acid sequences that code for the same amino acid sequence. A polynucleotide encoding an antibody (or a complete nucleic acid molecule) may be optimized for expression of the antibody. For example, codon optimization of the nucleotide sequence can be used to improve the efficiency of translation in an expression system for antibody production. In addition, the nucleic acid molecule may contain heterologous elements (i.e., elements that are not naturally present on the same nucleic acid molecule as the coding sequence of the antibody (heavy or light chain). For example, the nucleic acid molecule may contain a heterologous promoter, a heterologous enhancer, a heterologous UTR (e.g., for optimal translation / expression), a heterologous Poly-A-tail, etc.

[0107] A nucleic acid molecule is a molecule that contains a nucleic acid component. The term nucleic acid molecule usually refers to a DNA or RNA molecule. It is used interchangeably with the term "polynucleotide", i.e., a nucleic acid molecule can consist of a polynucleotide that codes for an antibody. Alternatively, a nucleic acid molecule can contain additional elements in addition to a polynucleotide that codes for an antibody. Typically, a nucleic acid molecule is a polymer that contains or consists of nucleotide monomers that are covalently linked to each other by sugar / phosphate backbone phosphodiester bonds. The term "nucleic acid molecule" also encompasses modified nucleic acid molecules, DNA or RNA molecules, such as base modifications, sugar modifications, backbone modifications, etc.

[0108] In general, nucleic acid molecules can be engineered to insert, delete or modify specific nucleic acid sequences. Such engineered changes include, but are not limited to, changes to introduce restriction sites, changes to correct codon usage, changes to add or optimize transcriptional and / or translational regulatory sequences, and the like. Nucleic acids can also be altered to modify the encoded amino acids. For example, it can be useful to introduce one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions, deletions, and / or insertions into the amino acid sequence of an antibody. Such point mutations can modify effector functions, antigen binding affinity, post-translational modifications, immunogenicity, etc., can introduce amino acids for attaching covalent groups (e.g., labels), or can introduce tags (e.g., for purification purposes). Alternatively, mutations in nucleic acid sequences can be "silent", i.e., not reflected in the amino acid sequence due to redundancy in the genetic code. In general, mutations can be introduced at specific sites or can be introduced randomly and through selection (e.g., molecular evolution). For example, one or more nucleic acids encoding either the light or heavy chains of an (exemplary) antibody can be randomly or directionally mutated to introduce different properties into the encoded amino acids. Such changes can be the result of an iterative process in which the original changes are retained and new changes at other nucleotide positions are introduced. Furthermore, changes achieved in independent steps can be combined.

[0109] In some embodiments, the polynucleotide (or (complete) nucleic acid molecule) encoding the antibody or antigen-binding fragment thereof may be codon-optimized. Various tools for codon optimization are known to the skilled artisan and are described, for example, in Ju Xin Chin, Bevan Kai-Sheng Chung, Dong-Yup Lee, Codon Optimization OnLine (COOL): a web-based multi-objective optimization platform for synthetic gene design, Bioinformatics, Volume 30, Issue 15, 1 August 2014, Pages 2210-2212; or Grote A, Hiller K, Scheer M, Munch R, Nortemann B, Hempel DC, Jahn D, JCat: a novel tool to adapt codon usage of a target gene to its potential expression host. Nucleic Acids Res. 2005 Jul 1;33(Web Server issue):W526-31; or, for example, in Genscript's OptimumGene TM algorithm (as described in US 2011 / 0081708 A1).

[0110] For example, a nucleic acid molecule of the present invention may comprise a nucleic acid sequence set forth in any one of SEQ ID NOs: 27-48; or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

[0111] Exemplary nucleic acid sequences encoding the CDRs, VH / VL or Fab sequences of exemplary antibodies of the invention are shown in Table 2 below. [Table 2]

[0112] Thus, the nucleic acid molecule may comprise: (i) the polynucleotide set forth in SEQ ID NO: 27 or 38 (or a sequence variant thereof); the polynucleotide set forth in SEQ ID NO: 28 or 39 (or a sequence variant thereof); the polynucleotide set forth in SEQ ID NO: 29 or 40 (or a sequence variant thereof); the polynucleotide set forth in SEQ ID NO: 30 or 41 (or a sequence variant thereof); (a) the polynucleotide set forth in SEQ ID NO: 31 or 32 (or a sequence variant thereof) or (b) the polynucleotide set forth in SEQ ID NO: 42 or 43 (or a sequence variant thereof); and the polynucleotide set forth in SEQ ID NO: 33 or 44 (or a sequence variant thereof); (ii) a polynucleotide set forth in SEQ ID NO: 34 or 45 (or a sequence variant thereof); and a polynucleotide set forth in SEQ ID NO: 35 or 46 (or a sequence variant thereof); or (iii) the polynucleotide set forth in SEQ ID NO: 36 or 47 (or a sequence variant thereof); and the polynucleotide set forth in SEQ ID NO: 37 or 48 (or a sequence variant thereof).

[0113] The present invention also provides a combination of a first and a second nucleic acid molecule, where the first nucleic acid molecule comprises a polynucleotide encoding a heavy chain of an antibody or antigen-binding fragment thereof of the present invention; and the second nucleic acid molecule comprises a polynucleotide encoding a corresponding light chain of the same antibody or the same antigen-binding fragment thereof. The above description of the (general) features of the nucleic acid molecules of the present invention also applies to the combined first and second nucleic acid molecules, as appropriate. Thus, one or both of the polynucleotides encoding the heavy and / or light chains of the antibody or antigen-binding fragment thereof may be codon-optimized. For example, the combination may comprise a nucleic acid sequence according to any one of SEQ ID NOs: 27-48; or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. In some embodiments, a combination of a first and a second nucleic acid molecule, wherein the first nucleic acid molecule comprises a polynucleotide encoding a heavy chain of an antibody or antigen-binding fragment thereof of the invention; and the second nucleic acid molecule comprises a polynucleotide encoding the corresponding light chain of the same antibody or antigen-binding fragment thereof, is as described below.

[0114] The present invention also provides a combination of a first nucleic acid molecule and a second nucleic acid molecule, - the first nucleic acid molecule comprises a polynucleotide encoding a heavy chain of an antibody or antigen-binding fragment thereof, the polynucleotide comprising (a) a nucleotide sequence set forth in SEQ ID NOs: 27, 28 and 29 (or sequence variants thereof); or (b) a nucleotide sequence set forth in SEQ ID NOs: 38, 39 and 40 (or sequence variants thereof); and - the second nucleic acid molecule comprises a polynucleotide encoding a light chain of the antibody or antigen-binding fragment thereof, the polynucleotide comprising (c) a nucleotide sequence set forth in SEQ ID NO: 30, 31 (or 32) and 33 (or sequence variants thereof), or (d) a nucleotide sequence set forth in SEQ ID NO: 41, 42 (or 43) and 44 (or sequence variants thereof).

[0115] Such a combination typically encodes an antibody or antigen-binding fragment thereof according to the invention as described above. Again, the above explanations regarding the (general) characteristics of the nucleic acid molecules of the invention also apply accordingly to the combined first and second nucleic acid molecules.

[0116] In some embodiments, - the first nucleic acid molecule comprises a polynucleotide comprising: (a) a nucleotide sequence set forth in SEQ ID NO: 34 (or a sequence variant thereof); or (b) a nucleotide sequence set forth in SEQ ID NO: 45 (or a sequence variant thereof), and - the second nucleic acid molecule comprises a polynucleotide comprising: (c) a nucleotide sequence set forth in SEQ ID NO: 35 (or a sequence variant thereof); or (d) a nucleotide sequence set forth in SEQ ID NO: 46 (or a sequence variant thereof).

[0117] In some embodiments, - the first nucleic acid molecule comprises a polynucleotide comprising: (a) a nucleotide sequence set forth in SEQ ID NO: 36 (or a sequence variant thereof); or (b) a nucleotide sequence set forth in SEQ ID NO: 47 (or a sequence variant thereof), and - the second nucleic acid molecule comprises a polynucleotide comprising: (c) a nucleotide sequence set forth in SEQ ID NO: 37 (or a sequence variant thereof); or (d) a nucleotide sequence set forth in SEQ ID NO: 48 (or a sequence variant thereof).

[0118] For the exemplified sequences as described herein, it is understood that such combinations of SEQ ID NOs corresponding to either antibody TT104 or antibody TT110 as shown in Table 2 above are preferred.

[0119] <vector> Further included within the scope of the present invention is a vector, such as an expression vector, which comprises a nucleic acid molecule according to the present invention. Typically, the vector comprises a nucleic acid molecule as described above.

[0120] The present invention also provides a combination of a first and a second vector, wherein the first vector comprises a first nucleic acid molecule as described above (for combinations of nucleic acid molecules) and the second vector comprises a second nucleic acid molecule as described above (for combinations of nucleic acid molecules).

[0121] A vector is usually a recombinant nucleic acid molecule, i.e. a nucleic acid molecule that does not occur in nature. Thus, a vector may contain heterologous elements (i.e. sequence elements that do not occur in nature). For example, a vector may contain a multiple cloning site, a heterologous promoter, a heterologous enhancer, a heterologous selection marker (to identify cells containing said vector compared to cells not containing said vector), etc. A vector in the context of the present invention is suitable to incorporate or carry a desired nucleic acid sequence. Such a vector may be a storage vector, an expression vector, a cloning vector, a transfer vector, etc. A storage vector is a vector that allows for convenient storage of a nucleic acid molecule. Thus, a vector may contain, for example, a sequence corresponding to a desired antibody (heavy and / or light chain) according to the invention. An expression vector may be used for the production of an expression product, such as RNA, e.g. mRNA, or a peptide, polypeptide or protein. For example, an expression vector may contain sequences necessary for the transcription of sequence extensions of the vector, such as a (heterologous) promoter sequence. A cloning vector is typically a vector that contains a cloning site that can be used to incorporate a nucleic acid sequence into the vector. The cloning vector may be, for example, a plasmid vector or a bacteriophage vector. The transfer vector is a vector suitable for transferring a nucleic acid molecule into a cell or organism, for example a viral vector. The vector in the context of the present invention may be, for example, an RNA vector or a DNA vector. For example, a vector in the sense of the present application comprises a cloning site, a selection marker such as an antibiotic resistance factor, and a sequence suitable for vector propagation, such as an origin of replication. The vector in the context of the present application may be a plasmid vector.

[0122] <cell> In further aspects, the present invention also provides a cell expressing an antibody or antigen-binding fragment thereof according to the invention; and / or a cell comprising a vector (or a combination of vectors) according to the invention.

[0123] Examples of such cells include, but are not limited to, eukaryotic cells, such as yeast cells, animal cells, or plant cells. Other examples of such cells include, but are not limited to, prokaryotic cells, such as E. coli. In some embodiments, the cell is a mammalian cell, such as a mammalian cell line. Examples include human cells, CHO cells, HEK293T cells, PER.C6 cells, NS0 cells, human hepatocytes, myeloma cells, or hybridoma cells.

[0124] The cell may be transfected with a vector according to the invention, for example an expression vector. The term "transfection" refers to the introduction of a nucleic acid molecule, such as a DNA or RNA (e.g., mRNA) molecule, into a cell, for example a eukaryotic or prokaryotic cell. In the context of the present invention, the term "transfection" includes any method known to those skilled in the art for introducing a nucleic acid molecule into a cell, such as a mammalian cell. Such methods include, for example, electroporation, lipofection, for example based on cationic lipids and / or liposomes, calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or cationic polymer-based transfection, such as DEAE-dextran or polyethyleneimine. In some embodiments, the introduction is non-viral.

[0125] Furthermore, the cells of the invention may be stably or transiently transfected with a vector according to the invention, e.g., to express an antibody according to the invention. In some embodiments, the cells are stably transfected with a vector according to the invention encoding an antibody according to the invention. In other embodiments, the cells are transiently transfected with a vector according to the invention encoding an antibody according to the invention.

[0126] Thus, the present invention also provides a recombinant host cell that heterologously expresses the antibody or antigen-binding fragment thereof of the present invention. For example, the cell may be of a different species than the antibody (e.g., a CHO cell expressing a human antibody). In some embodiments, the cell type of the cell does not naturally express the antibody (such as such). Furthermore, the host cell may impart post-translational modifications (PTMs; e.g., glycosylation) to the antibody that are not present in the native state. Such PTMs may result in functional differences (e.g., reduced immunogenicity). Thus, the antibody or antigen-binding fragment thereof of the present invention may have post-translational modifications that differ from naturally produced antibodies (e.g., antibodies of the human immune response).

[0127] <Antibody production> The antibody according to the present invention can be produced by any method known in the art.For example, the general methodology for producing monoclonal antibodies using hybridoma technology is known (Kohler, G. and Milstein, C., 1975; Kozbar et al. 1983).In some embodiments, the alternative EBV immortalization method described in WO2004 / 076677 is used.

[0128] In some embodiments, the methods described in WO2004 / 076677, incorporated herein by reference, are used. In this method, B cells producing the antibodies of the invention are transformed with EBV and polyclonal B cell activator. Additional stimulants of cell proliferation and differentiation may be added during the transformation step, optionally to further increase efficiency. These stimulants may be cytokines, such as IL-2 and IL-15. In one embodiment, IL-2 is added during the immortalization step to further increase the efficiency of immortalization, but its use is not essential. The immortalized B cells produced using these methods can then be cultured and antibodies isolated therefrom using methods known in the art.

[0129] Another exemplary method is described in WO2010 / 046775. In this method, plasma cells are cultured in microwell culture plates, in limited numbers, or as single plasma cells. Antibodies can be isolated from the plasma cell culture. In addition, RNA can be extracted from the plasma cell culture and PCR can be performed using methods known in the art. The VH and VL regions of the antibody can be amplified by RT-PCR (reverse transcriptase PCR), sequenced, and then cloned into an expression vector that is transfected into HEK293T cells or other host cells. Cloning of the nucleic acid into an expression vector, transfection of the host cell, culturing of the transfected host cell, and isolation of the produced antibody can be performed using any method known to those skilled in the art.

[0130] The antibodies can be further purified, if desired, using filtration, centrifugation, and various chromatographic methods, such as HPLC or affinity chromatography. Techniques for purifying antibodies, e.g., monoclonal antibodies, are known in the art, including techniques for producing pharmaceutical grade antibodies.

[0131] Standard techniques of molecular biology may be used to prepare DNA sequences encoding the antibodies of the present invention. The desired DNA sequence may be fully or partially synthesized using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may also be used as appropriate.

[0132] Any suitable host cell / vector system may be used for the expression of the DNA sequence encoding the antibody molecule of the present invention. Eukaryotic, e.g., mammalian host cell expression systems may be used for the production of antibody molecules, such as complete antibody molecules. Suitable mammalian host cells include, but are not limited to, CHO, HEK293T, PER.C6, NS0, myeloma or hybridoma cells. Prokaryotic, e.g., bacterial host cell expression systems may also be used for the production of antibody molecules, such as complete antibody molecules. Suitable bacterial host cells include, but are not limited to, E. coli cells.

[0133] The invention also provides a method for producing an antibody molecule according to the invention which comprises culturing a (heterologous) host cell containing a vector encoding a nucleic acid of the invention under conditions suitable for expression of protein from DNA encoding the antibody molecule of the invention and isolating the antibody molecule.

[0134] To produce an antibody containing both heavy and light chains, a cell line may be transfected with two vectors, a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide, or a single vector containing sequences encoding both light and heavy chain polypeptides may be used.

[0135] Antibodies according to the invention may be produced by (i) expressing a nucleic acid sequence according to the invention in a host cell, for example by use of a vector according to the invention, and (ii) isolating the expressed antibody product. In addition, the method may include (iii) purifying the isolated antibody. Transformed B cells and cultured plasma cells can be screened for those that produce antibodies with the desired specificity or function.

[0136] The screening step may be performed by any immunoassay, such as ELISA, staining of tissues or cells (including transfected cells), neutralization assays, or any of the many other methods known in the art for identifying a desired specificity or function. The assay may select based on simple recognition of one or more antigens, or may further select based on a desired function, such as the ability to select neutralizing antibodies as well as antigen-binding antibodies, to select antibodies that can alter the properties of the target cells, such as signaling cascades, shape, growth rate, ability to affect other cells, response to influences by other cells or other reagents or changes in conditions, differentiation state, etc.

[0137] Individual transformed B cell clones may then be produced from the positive transformed B cell cultures. The cloning step to separate individual clones from the mixture of positive cells can be performed using limiting dilution, micromanipulation, single cell deposition by cell sorting, or other methods known in the art.

[0138] Nucleic acids from the cultured plasma cells can be isolated, cloned, and expressed in HEK293T cells or other known host cells using methods known in the art.

[0139] The immortalized B cell clones or transfected host cells of the invention can be used in a variety of ways, e.g., as a source of monoclonal antibodies, as a source of nucleic acid (DNA or mRNA) encoding a monoclonal antibody of interest, for research purposes, etc.

[0140] The invention also provides compositions comprising immortalized B memory cells or transfected host cells that produce antibodies according to the invention.

[0141] The immortalized B cell clones or cultured plasma cells of the invention may also be used as a nucleic acid source for cloning of antibody genes for subsequent recombinant expression. Expression from recombinant sources may be more common for pharmaceutical purposes than expression from, for example, B cells or hybridomas, for reasons of stability, reproducibility, ease of culture, etc.

[0142] Therefore, the present invention also provides a method for preparing a recombinant cell comprising the steps of: (i) obtaining one or more nucleic acids (e.g., heavy and / or light chain mRNA) encoding an antibody of interest from a B cell clone or cultured plasma cell; (ii) inserting the nucleic acid(s) into an expression vector; and (iii) transfecting the vector into a (heterologous) host cell to allow expression of the antibody of interest in the host cell.

[0143] Similarly, the invention also provides a method for preparing a recombinant cell comprising the steps of: (i) determining the sequence of a nucleic acid from a B cell clone or cultured plasma cell that encodes an antibody of interest; and (ii) using the sequence information from step (i) to prepare a nucleic acid for insertion into a host cell to enable expression of the antibody of interest in the host cell. The nucleic acid may, but need not, be manipulated between steps (i) and (ii) to introduce restriction sites, alter codon usage, and / or optimize transcriptional and / or translational regulatory sequences.

[0144] Furthermore, the present invention also provides a method of preparing a transfected host cell comprising the step of transfecting the host cell with one or more nucleic acids encoding an antibody of interest, wherein the nucleic acids are nucleic acids derived from an immortalized B cell clone or a cultured plasma cell of the present invention. Thus, the procedures of first preparing the nucleic acid and then transfecting the host cell with it can be performed at different times in different places (e.g., in different countries) by different people.

[0145] Such recombinant cells of the invention can be used for expression and culture purposes. They are particularly useful for expressing antibodies for large-scale pharmaceutical production. They can also be used as an active ingredient in pharmaceutical compositions. Any suitable culture technique can be used, including but not limited to static culture, roller bottle culture, ascites, hollow fiber bioreactor cartridges, modular mini-fermenters, stirred tanks, microcarrier culture, ceramic core perfusion, etc.

[0146] Methods for obtaining and sequencing immunoglobulin genes from B cells or plasma cells are well known in the art (see, for example, Chapter 4 of Kuby Immunology, 4th edition, 2000).

[0147] The transfected host cells may be eukaryotic cells including yeast and animal cells, particularly mammalian cells (e.g., human cells such as CHO cells, NS0 cells, PER.C6 cells or HKB-11 cells, myeloma cells, or human hepatocytes), as well as plant cells. In some embodiments, the transfected host cells are mammalian cells, such as human cells. In some embodiments, the expression host is capable of glycosylation of the antibodies of the invention, particularly with carbohydrate structures that are not themselves immunogenic in humans. In some embodiments, the transfected host cells may be capable of growth in serum-free medium. In further embodiments, the transfected host cells may be capable of growth in culture without the presence of animal-derived products. The transfected host cells may be cultured to obtain cell lines.

[0148] The present invention also provides a method for preparing one or more nucleic acid molecules (e.g., heavy and light chain genes) encoding an antibody of interest, comprising the steps of: (i) preparing an immortalized B cell clone or culturing plasma cells according to the present invention; (ii) obtaining a nucleic acid encoding the antibody of interest from the B cell clone or cultured plasma cells. The present invention further provides a method for obtaining a nucleic acid sequence encoding an antibody of interest, comprising the steps of: (i) preparing an immortalized B cell clone or culturing plasma cells according to the present invention; (ii) sequencing a nucleic acid encoding the antibody of interest from the B cell clone or cultured plasma cells.

[0149] The invention further provides a method for preparing a nucleic acid molecule encoding an antibody of interest, comprising the step of obtaining nucleic acid obtained from a transformed B cell clone or cultured plasma cell of the invention. Thus, the steps of first obtaining the B cell clone or cultured plasma cell and then obtaining nucleic acid from the B cell clone or cultured plasma cell can be performed at different times by different people in different places (e.g. in different countries).

[0150] The present invention also includes methods for preparing an antibody according to the invention (e.g., for pharmaceutical use), comprising the steps of: (i) obtaining and / or sequencing one or more nucleic acids (e.g., heavy and light chain genes) from a selected B cell clone or cultured plasma cell expressing the antibody of interest; (ii) inserting the nucleic acid(s) or using the sequence of the nucleic acid(s) to prepare an expression vector; (iii) transfecting a host cell capable of expressing the antibody of interest; (iv) culturing or subculturing the transfected host cell under conditions in which the antibody of interest is expressed; and, optionally, (v) purifying the antibody of interest.

[0151] The present invention also provides a method for preparing an antibody of interest, comprising the steps of culturing or subculturing a transfected host cell population, e.g. a stably transfected host cell population, under conditions in which the antibody of interest is expressed, and optionally purifying the antibody of interest, said transfected host cell population having been prepared by (i) providing a nucleic acid encoding a selected antibody of interest produced by a B cell clone or cultured plasma cells prepared as described above, (ii) inserting the nucleic acid into an expression vector, (iii) transfecting the vector in a host cell capable of expressing the antibody of interest, and (iv) culturing or subculturing the transfected host cell comprising the inserted nucleic acid to produce the antibody of interest. Thus, the steps of first preparing a recombinant host cell and then culturing it to express the antibody can be performed at entirely different times by different people in different places (e.g. different countries).

[0152] <Pharmaceutical Composition> The present invention also provides pharmaceutical compositions comprising one or more of the following: (i) an antibody or antigen-binding fragment thereof of the present invention; (ii) a nucleic acid or combination of nucleic acids of the invention; (iii) a vector or combination of vectors of the invention; and / or (iv) A cell expressing an antibody according to the invention or comprising a vector according to the invention. and optionally a pharma- ceutically acceptable excipient, diluent or carrier.

[0153] In other words, the present invention also provides a pharmaceutical composition comprising an antibody according to the present invention, a nucleic acid according to the present invention, a vector according to the present invention and / or a cell according to the present invention.

[0154] The pharmaceutical composition may also optionally include a pharma- ceutically acceptable carrier, diluent and / or excipient. The carrier or excipient may facilitate administration, but should not itself induce the production of antibodies harmful to the individual receiving the composition. It should also not be toxic. Suitable carriers may be large, slowly metabolized macromolecules, such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles. In some embodiments, the pharma-ceutically acceptable carrier, diluent and / or excipient in the pharmaceutical composition according to the present invention is not an active ingredient with respect to Clostridium tetani or tetanus infection.

[0155] Pharmaceutically acceptable salts can be used, for example inorganic acid salts such as hydrochlorides, hydrobromides, phosphates and sulfates, or organic acid salts such as acetates, propionates, malonates and benzoates.

[0156] The pharma- ceutically acceptable carrier in the pharmaceutical composition may contain additional liquids such as water, saline, glycerol, ethanol, etc. In addition, auxiliary substances such as wetting agents or emulsifying agents or pH buffering substances may be present in such compositions. Such carriers allow the pharmaceutical composition to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., and ingested by subjects.

[0157] The pharmaceutical composition of the present invention may be prepared in various forms. For example, the composition may be prepared as an injection, either as a liquid solution or suspension. Solid forms suitable for dissolution or suspension in a liquid vehicle prior to injection can also be prepared (e.g., lyophilized compositions similar to Synagis™ and Herceptin®, for reconstitution in sterile water containing a preservative). The composition may be prepared for topical administration, for example, as an ointment, cream or powder. The composition may be prepared for oral administration, for example, as a tablet or capsule, as a spray, or as a syrup (optionally flavored). The composition may be prepared for pulmonary administration, for example, using a fine powder or spray, as an inhaler. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, aural or ocular administration, for example, as a drop. The composition may be in the form of a kit, designed to be reconstituted just prior to administration to a subject. For example, a lyophilized antibody may be provided in kit form with sterile water or a sterile buffer.

[0158] In some embodiments, the (only) active ingredient in the composition is the antibody according to the present invention. Therefore, it may be easily degraded in the digestive tract. Therefore, when the composition is administered by a route that uses the digestive tract, the composition may contain an agent that protects the antibody from degradation but releases the antibody when absorbed from the digestive tract.

[0159] A thorough discussion of pharma- ceutically acceptable carriers is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472.

[0160] The pharmaceutical compositions of the invention generally have a pH between 5.5 and 8.5, and in some embodiments, this may be between 6 and 8, for example about 7. The pH may be maintained by the use of a buffering agent. The compositions may be sterile and / or pyrogen-free. The compositions may be isotonic for humans. In some embodiments, the pharmaceutical compositions of the invention are supplied in a sealed container.

[0161] The scope of the present invention includes compositions that exist in several dosage forms; including, but not limited to, forms suitable for parenteral administration, such as injection or infusion, for example, by bolus injection or continuous infusion. If the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and may contain formulating agents such as suspending agents, preservatives, stabilizers and / or dispersants. Alternatively, the antibody may be in a dry form, to be reconstituted with an appropriate sterile liquid before use.

[0162] Vehicle is typically understood to be a material suitable for storing, transporting, and / or administering a pharma- ceutical active compound, particularly a compound such as an antibody according to the present invention.For example, the vehicle may be a physiologically acceptable liquid suitable for storing, transporting, and / or administering a pharma- ceutical active compound, particularly an antibody according to the present invention.Once formulated, the composition of the present invention can be administered directly to a subject.In some embodiments, the composition is adapted for administration to a mammalian, e.g., a human subject.

[0163] The pharmaceutical composition of the present invention may be administered by any number of routes, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, topical, subcutaneous, nasal, enteral, sublingual, vaginal or rectal routes. In some embodiments, the pharmaceutical composition may be administered to the central nervous system. Thus, it may be administered, for example, via intrathecal, intraventricular, intracerebral, epidural, nasal, intranasal or perianal routes of administration. Hyposprays may also be used to administer the pharmaceutical composition of the present invention. Optionally, the pharmaceutical composition may be prepared for oral administration, e.g., as tablets, capsules, etc., for topical administration, or for injection, e.g., as a liquid solution or suspension. In some embodiments, the pharmaceutical composition is an injection. Solid forms suitable for solution or suspension in a liquid vehicle prior to injection are also included, e.g., the pharmaceutical composition may be in a lyophilized form.

[0164] For injection, e.g., intravenous, cutaneous, subcutaneous, or into the affected area, the active ingredient may be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art are well able to prepare suitable solutions using isotonic vehicles, e.g., sodium chloride injection, Ringer's injection, lactated Ringer's injection, and the like. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as necessary. Whether it is an antibody, peptide, nucleic acid molecule, or other pharma- ceutical useful compound according to the invention that is administered to an individual, it is usually administered in an "effective amount," e.g., a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be), which is sufficient to show benefit to the individual. The actual amount administered, the rate and time course of administration will depend on the nature and severity of what is being treated. For injection, the pharmaceutical composition according to the invention may be provided, e.g., in a pre-filled syringe.

[0165] The pharmaceutical composition of the present invention as defined above can also be orally administered in any orally acceptable dosage form, including, but not limited to, capsules, tablets, aqueous suspensions or solutions. For oral tablets, commonly used carriers include lactose and cornstarch. Lubricants such as magnesium stearate are also commonly added. For oral administration in capsule form, useful diluents include lactose and dried cornstarch. When an aqueous suspension is required for oral administration, the active ingredient, i.e., the transporter cargo conjugate molecule of the present invention as defined above, is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents can also be added.

[0166] The pharmaceutical composition of the present invention may also be administered locally, especially when the area or organ to be treated includes an area or organ that is easily accessible by topical application, such as an accessible epithelial tissue. Suitable topical formulations are easily prepared for each of these areas or organs. For topical application, the pharmaceutical composition of the present invention may be formulated into a suitable ointment that contains the pharmaceutical composition of the present invention, particularly its components as defined above, suspended or dissolved in one or more carriers. Carriers for topical administration include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax and water. Alternatively, the pharmaceutical composition of the present invention may be formulated into a suitable lotion or cream. In the context of the present invention, suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

[0167] The administration treatment can be a single dose schedule or a multiple dose schedule. In particular, the pharmaceutical composition can be provided as a single dose product. In some embodiments, the amount of antibody in the pharmaceutical composition does not exceed 200 mg, for example does not exceed 100 mg or 50 mg, especially when provided as a single dose product.

[0168] In the case of a single administration, for example daily, weekly or monthly administration, the amount of the antibody in the pharmaceutical composition according to the present invention does not exceed 1 g or 500 mg. In some embodiments, for a single administration, the amount of the antibody in the pharmaceutical composition according to the present invention may not exceed 200 mg, or 100 mg. For example, for a single administration, the amount of the antibody in the pharmaceutical composition according to the present invention may not exceed 50 mg.

[0169] A pharmaceutical composition typically contains an "effective" amount of one or more antibodies of the invention, i.e., an amount sufficient to treat, ameliorate, attenuate, alleviate or prevent a desired disease or condition, or an amount sufficient to exhibit a detectable therapeutic effect. A therapeutic effect also includes the reduction or attenuation of pathogenic potency or physical symptoms. The precise effective amount for any particular subject will depend on the subject's size, weight, health, the nature and extent of the pathology, and the therapeutic agent or combination of therapeutic agents selected for administration. The effective amount for a given situation will be determined by routine experimentation and is within the judgment of the clinician. For purposes of the present invention, an effective amount can generally be about 0.005 to about 100 mg / kg, e.g., about 0.0075 to about 50 mg / kg or about 0.01 to about 10 mg / kg. In some embodiments, an effective amount will be about 0.02 to about 5 mg / kg of an antibody of the invention (e.g., the amount of antibody in a pharmaceutical composition) relative to the body weight (e.g., in kg) of the individual to be administered.

[0170] Furthermore, the pharmaceutical composition according to the invention may comprise an additional active ingredient, which may be a further antibody or a non-antibody component. Thus, the pharmaceutical composition according to the invention may comprise one or more additional active ingredients. In some embodiments, the pharmaceutical composition comprises two different antibodies or antigen-binding fragments, in particular two different antibodies or antigen-binding fragments that (specifically) bind to tetanus toxin. In some embodiments, each of the two different antibodies or antigen-binding fragments is an antibody according to the invention as described above.

[0171] The antibody according to the invention can be present in the same pharmaceutical composition as an additional active ingredient (e.g., a second antibody as described above), or the antibody according to the invention is included in a first pharmaceutical composition and the additional active ingredient (e.g., a second antibody as described above) is included in a second pharmaceutical composition different from the first pharmaceutical composition. Thus, when one or more additional active ingredients are envisaged, each additional active ingredient (e.g., a second antibody as described above) and the antibody according to the invention may be included in different pharmaceutical compositions. Such different pharmaceutical compositions can be administered in combination / simultaneously or at different times or different locations (e.g., different parts of the body).

[0172] Thus, the present invention also provides a combination of two different antibodies or antigen-binding fragments thereof, wherein each of the two different antibodies or antigen-binding fragments thereof is an antibody according to the invention as described above, preferably an antibody for use in medicine, as described in more detail below.

[0173] Furthermore, the present invention also provides a kit of parts comprising two different antibodies or antigen-binding fragments thereof, wherein each of the two different antibodies or antigen-binding fragments thereof is an antibody according to the present invention as described above. In the kit of parts, the two different antibodies may be provided in different containers (e.g. in different pharmaceutical compositions).

[0174] In particular, for the above mentioned pharmaceutical compositions, combinations and kits of parts, of the two different antibodies or antigen-binding fragments thereof of the invention, the first antibody or antigen-binding fragment thereof may comprise the heavy chain CDR1 sequence set forth in SEQ ID NO: 1, the heavy chain CDR2 sequence set forth in SEQ ID NO: 2, the heavy chain CDR3 sequence set forth in SEQ ID NO: 3, the light chain CDR1 sequence set forth in SEQ ID NO: 4, the light chain CDR2 sequence set forth in SEQ ID NO: 5 or 6, and the light chain CDR3 sequence set forth in SEQ ID NO: 7; and the second antibody or antigen-binding fragment thereof may comprise the heavy chain CDR1 sequence set forth in SEQ ID NO: 12, the heavy chain CDR2 sequence set forth in SEQ ID NO: 13, the heavy chain CDR3 sequence set forth in SEQ ID NO: 14, the light chain CDR1 sequence set forth in SEQ ID NO: 15, the light chain CDR2 sequence set forth in SEQ ID NO: 16 or 17, and the light chain CDR3 sequence set forth in SEQ ID NO: 18.

[0175] In some embodiments, the first antibody or antigen-binding fragment may comprise a VH sequence as set forth in SEQ ID NO:8, or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity (e.g., containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations); and a VL sequence as set forth in SEQ ID NO:10, or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity (e.g., containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations), thereby maintaining the CDR sequences defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO:4, SEQ ID NO:5 or 6, and SEQ ID NO:7, respectively). The second antibody or antigen-binding fragment may then comprise the VH sequence set forth in SEQ ID NO: 19, or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity (e.g., containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations); and the VL sequence set forth in SEQ ID NO: 20, or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity (e.g., containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations). This allows the CDR sequences defined above to be maintained (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 or 17, and SEQ ID NO: 18, respectively).

[0176] The antibody according to the invention and an additional active ingredient (e.g., the second antibody described above) may provide an additive therapeutic effect, such as a synergistic therapeutic effect. The term "synergistic effect" is used to describe a combined effect of two or more active agents that is greater than the sum of the individual effects of each active agent. Thus, when the combined effect of two or more agents results in a "synergistic inhibition" of an activity or process, it is intended that the inhibition of the activity or process is greater than the sum of the inhibitory effects of each active agent. The term "synergistic therapeutic effect" refers to a therapeutic effect observed with the combination of two or more therapies, where the therapeutic effect (measured by several parameters) is greater than the sum of the individual therapeutic effects observed with each of the therapies individually.

[0177] In some embodiments, a pharmaceutical composition according to the invention may not comprise additional active ingredients (in addition to the antibody of the invention or the respective nucleic acid, vector or cell described above).

[0178] In some embodiments, a composition of the invention may comprise an antibody of the invention, where the antibody may comprise at least 50% by weight (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) of the total protein in the composition. In a composition of the invention, the antibody may be in a purified form.

[0179] The present invention also provides a method for preparing a pharmaceutical composition comprising the steps of: (i) preparing an antibody of the present invention; and (ii) admixing the purified antibody with one or more pharma- ceutically acceptable excipients, diluents or carriers.

[0180] In other embodiments, a method for preparing a pharmaceutical composition comprises admixing an antibody with one or more pharma- ceutically acceptable carriers, wherein the antibody is a monoclonal antibody obtained from the transformed B cells or cultured plasma cells of the invention.

[0181] Instead of delivering antibodies or B cells for therapeutic purposes, a nucleic acid (typically DNA) encoding a monoclonal antibody of interest derived from B cells or cultured plasma cells can be delivered to a subject, where the nucleic acid can be expressed in situ within the subject to provide the desired therapeutic effect. Suitable gene therapy and nucleic acid transfer vectors are known in the art.

[0182] Pharmaceutical compositions may also contain antimicrobial agents, especially when packaged in multi-dose form. They may also contain detergents, such as Tweens (polysorbates), e.g., Tween 80. Detergents are generally present at low levels, e.g., less than 0.01%. The compositions may also contain sodium salts (e.g., sodium chloride) to provide tonicity. For example, a NaCl concentration of 10±2 mg / ml is typical.

[0183] Additionally, pharmaceutical compositions, particularly if lyophilized or containing reconstituted material from lyophilized material, may contain, for example, a sugar alcohol (e.g., mannitol) or a disaccharide (e.g., sucrose or trehalose) at around 15-30 mg / ml (e.g., 25 mg / ml). The pH of the composition for lyophilization can be adjusted to 5-8, or 5.5-7, or around 6.1 prior to lyophilization.

[0184] Compositions of the invention may also include one or more immunomodulatory agents. In some embodiments, the one or more immunomodulatory agents include an adjuvant.

[0185] <Medical procedures and other uses> In a further aspect, the present invention provides the use of an antibody or antigen-binding fragment thereof according to the invention, a nucleic acid molecule (or combination of nucleic acid molecules) according to the invention, a vector (or combination of vectors) according to the invention, a cell according to the invention, a pharmaceutical composition according to the invention, a combination according to the invention or a kit of parts according to the invention as a medicament. In particular, an antibody or antigen-binding fragment thereof according to the invention, a nucleic acid molecule (or combination of nucleic acid molecules) according to the invention, a vector (or combination of vectors) according to the invention, a cell according to the invention, a pharmaceutical composition according to the invention, a combination according to the invention or a kit of parts according to the invention may be used for the prevention and / or treatment of Clostridium tetani infection or tetanus.

[0186] Thus, the present invention also provides a method of ameliorating or alleviating Clostridium tetani infection or tetanus or reducing the risk of Clostridium tetani infection or tetanus, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to the invention, a nucleic acid molecule (or combination of nucleic acid molecules) according to the invention, a vector (or combination of vectors) according to the invention, a cell according to the invention, or a pharmaceutical composition according to the invention to a subject in need thereof. Furthermore, the present invention also provides the use of an antibody or antigen-binding fragment thereof according to the invention, a nucleic acid molecule (or combination of nucleic acid molecules) according to the invention, a vector (or combination of vectors) according to the invention, a cell according to the invention, or a pharmaceutical composition according to the invention in the manufacture of a medicament for the prevention, treatment or attenuation of Clostridium tetani infection or tetanus.

[0187] Prevention of Clostridium tetani infection or tetanus refers in particular to a preventive setting where the subject is not diagnosed with Clostridium tetani infection or tetanus (no diagnosis was made or the diagnosis was negative) and / or the subject does not show symptoms of Clostridium tetani infection or tetanus.In contrast, in a therapeutic setting, the subject is typically diagnosed with Clostridium tetani infection or tetanus and / or shows symptoms of Clostridium tetani infection or tetanus.Note that the terms "treatment" and "therapy" / "therapeutic" of Clostridium tetani infection or tetanus include not only attenuation / reduction of Clostridium tetani infection or tetanus and / or related symptoms, but also (complete) cure.

[0188] In some embodiments, the subject may be a human. One method of confirming the effectiveness of the therapeutic treatment includes monitoring disease symptoms after administration of the composition of the present invention. The treatment may be a single dose schedule or a multiple dose schedule. In one embodiment, the antibody, antibody fragment, nucleic acid, vector, cell or composition according to the present invention is administered to a subject in need of such treatment. Such subjects include, but are not limited to, those at risk or susceptible to Clostridium tetani infection or tetanus, such as, for example, immunocompromised subjects.

[0189] The antibodies and fragments thereof according to the invention can also be used to diagnose Clostridium tetani infection or tetanus. The method of diagnosis can include contacting the antibody with a sample. Such a sample may be isolated from a subject, such as, for example, an isolated tissue sample taken from the nasal cavity, paranasal sinuses, salivary glands, lungs, liver, pancreas, kidneys, ears, eyes, placenta, gastrointestinal tract, heart, ovaries, pituitary gland, adrenal gland, thyroid gland, brain, skin, or blood, such as plasma or serum. For example, the antibody or antigen-binding fragment thereof may be contacted with an (isolated) blood sample (e.g., whole blood, plasma or serum). The method of diagnosis can also include detection of antigen / antibody complexes, in particular detection after contacting the antibody with the sample. Such a detection step is usually performed on the bench, i.e. without contacting the human or animal body. Examples of detection methods are well known to the skilled person and include, for example, ELISA (enzyme-linked immunosorbent assay). Thus, the diagnosis can be performed in vitro, for example, by using a sample isolated as described above (and an in vitro detection step of antigen / antibody complexes). Thus, the antibody or antigen-binding fragment thereof may be used for the (in vitro) diagnosis of Clostridium tetani infection or tetanus.

[0190] Thus, the antibody or antigen-binding fragment of the present invention can be used in an (in vitro) method for detecting an antigen, i.e., tetanus toxin. Similarly, the antibody or antigen-binding fragment of the present invention can be used in an (in vitro) method for binding a tetanus toxin target protein / antigen. To detect a tetanus toxin (antigen), the antibody can be contacted with an (isolated) sample (i.e., the sample to be tested for the presence of the antigen). Specific binding of the antibody to the antigen (tetanus toxin) results in the formation of an antibody / antigen complex, which can be easily detected by methods known in the art.

[0191] Such detection methods may be used in an (in vitro) diagnostic context (with samples isolated from the human or animal body), but also for testing other (e.g. production / manufacturing) samples, such as vaccine samples. Thus, the antibodies, antibody fragments or variants thereof according to the invention may also be used in a non-therapeutic / non-diagnostic context, for example in vaccine development or production. The invention therefore also provides the use of the antibodies or antigen-binding fragments thereof of the invention for testing vaccines, in particular whether the antigen (i.e. the desired antigen contained in the vaccine, such as tetanus toxin) is properly produced and / or folded (and / or in the correct conformation). Thus, the antibodies may be used for monitoring the production of vaccines with the desired immunogenicity. For this purpose, the antibodies may be contacted with the vaccine, for example as described above. Furthermore, the invention also encompasses the use of the antibodies or antigen-binding fragments thereof of the invention for monitoring the quality of anti-tetanus vaccines, by checking whether the vaccine contains the desired antigen, for example tetanus toxin, or a fragment or variant thereof. More specifically, the antibodies may be used for checking the conformation of the antigen or its epitope in the vaccine. Modified versions of the antigen (tetanus toxin) may also be tested with the antibodies of the invention, such as fragments and mutants of tetanus toxin that are useful in vaccines. The attached figures are briefly described below, which serve to explain the invention in more detail, but are not intended to limit the subject matter of the invention in any way.

[0192] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows, for Example 1, the binding characteristics to tetanus toxoid (TT) of monoclonal antibodies isolated from donors vaccinated with TT.

[0193] (Example) Below are given specific examples illustrating various embodiments and aspects of the present invention. However, the present invention is not limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and practice the present invention. However, the present invention is not limited in scope by the exemplified embodiments, which are intended only as illustrations of single aspects of the invention, and methods that are functionally equivalent are within the scope of the present invention. Indeed, various modifications of the present invention in addition to those described herein will be readily apparent to those skilled in the art from the foregoing description, the accompanying figures, and the following examples. All such modifications are within the scope of the appended claims.

[0194] Example 1: Identification and characterization of human monoclonal antibodies TT104 and TT110 Peripheral blood samples were collected from donors who had undergone routine vaccination with tetanus toxoid (TT). Memory B cells were isolated by magnetic cell sorting using anti-CD19-PECy7 antibody and mouse anti-PE microbeads, followed by FACS sorting using Alexa Fluor 647-labeled goat anti-human IgG, Alexa Fluor 647-labeled goat anti-human IgM, and PE-labeled anti-human IgD. Sorted IgG memory B cells were immortalized with Epstein-Barr virus (EBV) and plated in single cell culture in the presence of CpG-DNA and irradiated PBMC-feeder cells (Traggiai E. et al., 2004, Nat Med 10(8): 871-5 and described in WO2004 / 076677).

[0195] Two weeks after immortalization, culture supernatants were tested for binding to TT by ELISA. Briefly, ELISA plates were coated with 1 μg / ml of recombinant TT. Plates were blocked with 1% BSA and incubated with titrated antibodies, followed by incubation with 1 / 500 alkaline phosphatase (AP)-labeled goat anti-human IgG. Plates were then washed, substrate (paranitrophenyl phosphate (p-NPP), Sigma) was added, and plates were read at 405 nm. The results are shown in Figure 1 and Table 3. These data indicate that several immortalized B cell clones producing TT-specific monoclonal antibodies were identified. The nucleotide sequences of the antibody V genes of 10 TT-specific monoclonal antibodies were determined and analyzed using the IMGT database (IMGT: http: / / www.imgt.org / ; see Lefranc, M.-P. et al. (2009) Nucleic Acids Res. 37, D1006-D1012). [Table 3]

[0196] Among the antibodies with the highest binding affinity, TT110 and TT104 were selected, and FABs of these antibodies were prepared.

[0197] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Brief description of the drawings]

[0198] [Figure 1] The binding characteristics of monoclonal antibodies isolated from tetanus toxoid (TT)-vaccinated donors are shown for Example 1.

Claims

(i) a heavy chain CDR1 sequence set forth in SEQ ID NO: 1, a heavy chain CDR2 sequence set forth in SEQ ID NO: 2, a heavy chain CDR3 sequence set forth in SEQ ID NO: 3, a light chain CDR1 sequence set forth in SEQ ID NO: 4, a light chain CDR2 sequence set forth in SEQ ID NO: 5 or 6, and a light chain CDR3 sequence set forth in SEQ ID NO: 7; or (ii) a heavy chain CDR1 sequence set forth in SEQ ID NO: 12, a heavy chain CDR2 sequence set forth in SEQ ID NO: 13, a heavy chain CDR3 sequence set forth in SEQ ID NO: 14, a light chain CDR1 sequence set forth in SEQ ID NO: 15, a light chain CDR2 sequence set forth in SEQ ID NO: 16 or 17, and a light chain CDR3 sequence set forth in SEQ ID NO: 18; An antibody or antigen-binding fragment thereof that binds to tetanus toxin, comprising:

2. The antibody or antigen-binding fragment thereof (i) a heavy chain variable region comprising an amino acid sequence having at least 90% identity to SEQ ID NO:8, and a light chain variable region comprising an amino acid sequence having at least 90% identity to SEQ ID NO:9; or (ii) a heavy chain variable region comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 19, and a light chain variable region comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 20; The antibody or antigen-binding fragment thereof of claim 1, comprising:

3. The antibody or antigen-binding fragment thereof (i) a heavy chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID NO:8, and a light chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID NO:9; or (ii) a heavy chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 19, and a light chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 20; The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising:

4. The antibody or antigen-binding fragment thereof (i) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9; or (ii) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 19, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 20; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising:

5. The antibody or antigen-binding fragment thereof (i) a heavy chain comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 10, and a light chain comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 11; or (ii) a heavy chain comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 21, and a light chain comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 22; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising:

6. The antibody or antigen-binding fragment thereof (i) a heavy chain comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 10, and a light chain comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 11; or (ii) a heavy chain comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 21, and a light chain comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 22; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, comprising:

7. The antibody or antigen-binding fragment thereof (i) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 10, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 11; or (ii) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 21, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 22; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, comprising:

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof is a human antibody, a monoclonal antibody, and / or a purified antibody.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody comprises an Fc portion.

10. The antibody of any one of claims 1 to 9, wherein the antibody is of the IgG type, optionally the antibody is of the IgG1 type.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antibody or antigen-binding fragment thereof is Fab, Fab', F(ab')2, Fv or scFv.

12. A nucleic acid molecule comprising a polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 11, wherein optionally the polynucleotide is codon-optimized.

13. 13. The nucleic acid molecule of claim 12, comprising a nucleic acid sequence set forth in any one of SEQ ID NOs: 27-48; or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

14. A combination of a first and a second nucleic acid molecule, wherein the first nucleic acid molecule comprises a polynucleotide encoding a heavy chain of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11; and the second nucleic acid molecule comprises a polynucleotide encoding a corresponding light chain of the antibody or antigen-binding fragment thereof, optionally wherein the polynucleotide encoding the heavy chain and / or the polynucleotide encoding the light chain is codon-optimized.

15. 15. A combination of nucleic acid molecules according to claim 14, comprising a nucleic acid sequence set forth in any one of SEQ ID NOs: 27 to 48; or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

16. A vector comprising a nucleic acid molecule according to claim 12 or 13, or a combination of nucleic acid molecules according to claim 14 or 15.

17. A combination of a first vector and a second vector, wherein the first vector comprises a first nucleic acid molecule as defined in claim 14 or 15, and the second vector comprises a corresponding second nucleic acid molecule as defined in claim 14 or 15.

18. A cell expressing the antibody or antigen-binding fragment thereof of any one of claims 1 to 11, or comprising the vector of claim 16 or 17.

19. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, a nucleic acid according to claim 12 or 13, a combination of nucleic acids according to claim 14 or 15, a vector according to claim 16, a combination of vectors according to claim 17, or a cell according to claim 18, and optionally a pharmaceutically acceptable excipient, diluent or carrier.

20. An antibody or antigen-binding fragment thereof described in any one of claims 1 to 11, a nucleic acid described in claim 12 or 13, a combination of nucleic acids described in claim 14 or 15, a vector described in claim 16, a combination of vectors described in claim 17, or a cell described in claim 18, or a pharmaceutical composition described in claim 19, in the prevention or treatment of infection with Clostridium tetani or tetanus.

21. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 in the (in vitro) diagnosis of Clostridium tetani or tetanus infection.

22. 12. Use of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 11 for monitoring the quality of an anti-tetanus vaccine, wherein the quality of the anti-tetanus vaccine is monitored by confirming the conformation of the antigen or its epitope contained in the vaccine.

23. 20. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the nucleic acid according to claim 12 or 13, the combination of nucleic acids according to claim 14 or 15, the vector according to claim 16, the combination of vectors according to claim 17 or the cell according to claim 18, or the pharmaceutical composition according to claim 19, in the manufacture of a medicament for the prevention, treatment or attenuation of Clostridium tetani or tetanus infection.

Images